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CARDIOVASCULAR ANESTHESIA

The Effects of Heparin, Protamine, and Heparin/Protamine Reversal on Platelet Function Under Conditions of Arterial Shear Stress

Michael J. Griffin, MRCPI, FFARSI^*, Henry M. Rinder, MD, Brian R. Smith, MD, Jayne B. Tracey, Nancy S. Kriz, Conan K. Li, PhD, and Christine S. Rinder, MD^*

Departments of *Anesthesiology, Laboratory Medicine, Internal Medicine, and Pediatrics, Yale University School of Medicine and Yale-New Haven Hospital, New Haven, Connecticut; and Xylum Corporation, Scarsdale, New York

Address correspondence and reprint requests to Michael J. Griffin, Assistant Professor, Department of Anesthesiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8051. Address e-mail to michael.griffin{at}yale.edu

	Abstract

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Platelet dysfunction contributes to blood loss after cardiopulmonary bypass. This study examined the antiplatelet effects of heparin, protamine, and varying heparin/protamine ratios in an in vitrophysiologic model and further elucidated the mechanism of the antiplatelet and anticoagulant effects of protamine. We used the Clot Signature Analyzer (CSA^TM), a system that analyzes coagulation in flowing whole blood, to test two aspects of platelet function, with different concentrations of heparin and protamine, under conditions simulating arterial flow: collagen-induced thrombus formation (CITF) under moderate shear and high shear platelet activation, platelet hemostasis time (PHT). In addition, platelet aggregometry, celite activated clotting time (Hepcon^TM ACT), prothrombin time (PT), and partial thromboplastin time (PTT) were measured. Both PHT and the CITF were prolonged by heparin at 20 µg/mL, protamine at 20 and 40 µg/mL, and heparin/protamine ratios of 1:1 and 1:2, but not at 1:1.5. The Hepcon ACT was prolonged by heparin 20 µg/mL and protamine alone at 20 and 40 µg/mL, was normal at a ratio of 1:1, and was prolonged at 1:1.5 and 1:2. Protamine 80 µg/mL prolonged the PT and PTT. Dependency on thrombin, protein kinase C activation, and nonspecific charge effects were examined. The direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone prolonged the PHT and ACT, but not the CITF, whereas the polycationic molecules polyarginine and polylysine prolonged the CITF, but not the PHT. The effect of protamine on the PTT, but not PT, could be shortened by the addition of excess phospholipid. Therefore, heparin inhibits both high shear collagen-independent and moderate shear collagen-dependent platelet activation; however, the latter is not mediated by its antithrombin activity. Protamine’s antithrombin effect may explain its inhibition of platelet activation at high shear stress. Protamine’s nonspecific charge effects are more important for inhibiting moderate shear collagen-induced platelet activation.

Implications: This study suggests that protamine reversal of heparin’s antiplateleteffect occurs within a narrow window because of the direct antiplateleteffects of protamine. Antithrombin effects may explain the inhibition of shearactivation of platelets by both heparin and protamine. Nonspecific chargeeffects of protamine may explain the inhibition of collagen plateletactivation in the presence of medium shear.

	Introduction

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Platelet dysfunction contributes to blood loss after cardiopulmonary bypass and is partly mediated by the effects of extracorporeal circulation and the oxygenator (1). However, protamine, heparin, and the heparin/protamine complex also disrupt platelet function (2–4).

The specific antiplatelet effects of heparin at pharmacologic levels are well documented by in vitrostatic assays (4,5). Heparin binds to high-affinity platelet membrane sites (6,7), blocks von Willebrand factor (vWF)/platelet interaction at the glycoprotein (GP)-Ib receptor (8), and reduces platelet P selectin expression to agonists (9–12). Protamine is a highly cationic polypeptide, with 60% of its primary sequence accounted for by arginine. The protamine molecule has nonspecific charge-dependent effects and is also a potent activator of protein kinase C (PKC) (13). Protamine inhibits platelet function in vitro, but only at levels well above the clinical range (2).

Although prior work in static in vitrosystems provides a basis for understanding the clinical consequences of the administration of these drugs, little is known about the effects of heparin, protamine, and the heparin/protamine complex in clinically relevant concentrations on platelet function under shear conditions analogous to normal arterial blood flow. Within the dynamic arterial circulation, platelets use different activation pathways and receptor/ligand pairs compared with those studied in nonflow circumstances (14). These are highly dependent on the level of shear forces, which in small arteries and arterioles reaches levels of 1500–5000/s (14).

One of the original devices designed to study coagulation in a flow system was the hemostatometer, developed in the 1980s (15,16). This device required anticoagulated blood (15,16). Subsequent development led to devices, including the Clot Signature Analyzer (CSA^TM; Xylum Corp., Scarsdale, NY), that could analyze nonanticoagulated blood (16,17). The CSA tests platelet function under physiologic conditions of pressure (65 mm Hg) and shear (>1500/s), simulating small to medium arteries without anticoagulation (18,19). Platelet adhesion to damaged subendothelium in this shear range is dependent on binding to collagen-bound vWF and activation by exposed collagen.

The device has two channels, each of which is loaded with a 3-mL syringe of blood. In one CSA channel, flowing blood is exposed to a suspended collagen fibril; platelets are activated by and adhere to the collagen fibril. The time required for the developing platelet plug to occlude the channel assesses the platelet agonist response and the integrity of the platelet plug to arterial shear. This measure is termed the collagen-induced thrombus formation time (CITF) (Figs. 1A and 2B). Therefore, the CITF assesses platelet activation and aggregation and thrombus formation under conditions analogous to subendothelial collagen exposure after vessel injury. Blood flowing under similar conditions in the second CSA channel is subjected to a 0.15-mm side-wall puncture, allowing blood to exit at very high shear, >10,000/s, mimicking the conditions produced by an arterial wall puncture. Under these conditions, platelet adhesion is dependent on the binding of GP-Ib to the A1 binding site exposed by high shear on immobilized vWF. This results in shear-dependent signal transduction (14) and platelet activation by thrombin. The time required for platelets to completely occlude the puncture in the absence of exogenous agonist reflects this shear activation and is termed the platelet hemostasis time (PHT) (Figs. 1B, 1C, and 2A). The time required for the platelet plug, now protruding into the main capillary lumen, to activate the soluble coagulation cascade and completely obstruct the main capillary lumen is termed the clotting time (Fig. 2A).

View larger version (87K): [in this window] [in a new window]   Figure 1. A, A scanning electron micrograph of the collagen channel of the Clot Signature Analyzer (CSATM) after completion of the collagen-induced thrombus formation. Note the thick layer of platelet aggregates and fibrin mesh covering the surface and the bare fiber surface visible to the right. B, A scanning electron micrograph of the blood tube lumen at the punch site of the second channel of the CSA 2 min after punch formation. Note the characteristic morphology of fully spread activated adherent platelets to the left. Moving to the right near the entrance to the punch hole are numerous platelet aggregates with extensive pseudopods. C, A scanning electron micrograph of a transverse section through the punch site of the CSA after completion of the platelet-mediated hemostasis time, i.e., punch closure caused by platelet plug formation. Extending into the lumen to the right is fibrin clot formation with trapped red blood cells, which partially occludes the lumen. (Micrographs provided by TJ Hoffmann. Reproduced with permission from Xylum Corporation.)

View larger version (28K): [in this window] [in a new window]   Figure 2. The Clot Signature Analyzer (CSATM) subjects unanticoagulated whole blood to conditions simulating arterial shear. Pressure in both capillary tubes is measured over time. A, In one capillary tube the time required, after a puncture in the tubing, to form a completely occlusive platelet plug reflects the platelets’ ability to activate in response to shear and is termed the platelet-mediated hemostasis time. This value is defined as time from puncture to time of 90% restoration of baseline tube pressure. The time required for the platelet plug, now protruding into the main capillary lumen, to activate the soluble coagulation cascade and completely obstruct the capillary lumen is termed the clotting time. B, In the second capillary tube, flowing blood is exposed to a collagen fibril, and the time to complete flow obstruction assesses the platelet agonist response and the integrity of the plug to arterial shear; this is termed the collagen-induced thrombus formation time. This value is defined as the time it takes to when the pressure in the collagen channel decreases to 50% of the initial run pressure.

	Methods

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After obtaining approval by the Yale University Human Investigation Committee and informed consent, we studied healthy volunteers. Any subject with a bleeding tendency (history of easy bruising) or who had ingested any medication known to interfere with platelet function (aspirin or nonsteroidal antiinflammatory drugs) within the previous 10 days was excluded. Whole-blood samples were drawn into syringes containing the test solution or an equal volume of diluent, phosphate buffered saline (PBS). Blood was mixed and incubated for 2 min, then it was subjected to assay on the CSA, Hepcon^TM ACT (International Technidyne Inc., Edison, NJ), and whole-blood platelet aggregometry (Chronolog^TM; Havertown, PA).

Concentrations of heparin used were based on previously published work describing concentrations obtained in vivo after a dose of 300 U/kg (20). Protamine concentrations were determined after the previously demonstrated in vitro effect of 20 µg/mL protamine on the ACT in the protamine response test (21). The corresponding protamine dose in a given patient may be calculated by using the estimated blood volume because the initial volume of distribution of protamine is close to the intravascular volume (21).

Unfractionated porcine heparin (1000 U/mL) and protamine sulfate (10 mg/mL) were obtained from Elkins-Sinn, Cherry Hill, NJ. Three heparin/protamine concentration ratios were examined with both the CSA and the ACT and compared with simultaneous PBS controls in 12 volunteers: 1) a 1:1 ratio (40 µg/mL [4 U/mL] heparin and 40 µg/mL protamine, corresponding to the expected blood concentrations after the administration of 3 mg/kg of both drugs); 2) a 1:1.5 ratio (40 µg/mL heparin and 60 µg/mL protamine, corresponding to 3 and 4.5 mg/kg doses, respectively); and 3) a 1:2 ratio (40 µg/mL heparin and 80 µg/mL protamine, corresponding to 3 and 6 mg/kg doses, respectively). Heparin alone was studied with the CSA at final concentrations of 4 and 20 µg/mL in 12 volunteers. Protamine alone was studied with both the CSA and the ACT at final concentrations of 4, 20, and 40 µg/mL in 12 volunteers.

The specific thrombin inhibitor PPACK was studied with the CSA at final concentrations of 0.1, 1, and 10 µM and compared with simultaneous paired PBS controls in blood from 10 volunteers. These PPACK concentrations were equivalent to heparin 4, 40, and 400 µg/mL in antithrombin activity (22).

Protamine is a potent activator of PKC, which is normally activated by arginine-rich proteins (13). We compared the platelet effects of the PKC activator polycationic PA and the similarly highly cationic but non-PKC-activating compound PL (13). Whole blood samples from 10 volunteers were studied in the CSA and the ACT after incubation with either 100 nM PA or 100 nM PL simultaneously with PBS control samples.

Blood was drawn from seven volunteers into citrate anticoagulant (0.38% final concentration). Samples were studied by platelet aggregometry (Chronolog) after incubation for 2 min with PBS or protamine at concentrations of 4, 40, and 80 µg/mL, with heparin/protamine at 1:1, 1:1.5, and 1:2 ratios (concentrations as above), followed by aggregation at 37°C with collagen at a final concentration of 32 µg/mL. Aggregation was measured as the maximum change in light transmittance; all aggregation values were reported as a percentage of the PBS control value to compare studies between subjects.

Aliquots of citrate-anticoagulated blood were also spun for plasma, which was incubated with protamine alone at concentrations of 40 and 80 µg/mL or heparin/protamine at 1:1, 1:1.5, and 1:2 ratios (concentrations as above). Plasma was then examined on the MLA-1800 (International Laboratories, Pleasantville, NY) for PT and PTT. Prolonged PT and PTT values in the presence of protamine were then studied with the addition of excess phospholipid for correction.

The paired Student’s t-test was used to compare CSA platelet function parameters (PHT and CITF) and ACT in the presence of heparin, protamine, heparin/protamine, PA, PL, and PPACK with paired PBS controls. Similarly, the paired Student’s t-test was used to compare PT, PTT, and aggregometry slope and maximum amplitude in the presence of protamine or heparin/protamine with paired PBS control samples. A P value 0.05 was considered statistically significant.

	Results

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The CITF tests the platelet’s ability to generate an effective occlusive platelet plug in response to collagen at moderately high shear. The CITF was prolonged by protamine at 20 and 40 µg/mL, concentrations suitable for a 1:1 reversal of heparin at 2 and 4 U/mL, respectively (P < 0.05) (Fig. 3A), but not by small-dose protamine (4 µg/mL). By contrast, aggregometry in the presence of protamine did not show inhibition of collagen-induced platelet aggregation at concentrations up to 80 µg/mL (data not shown)—four times the minimum concentration that prolonged the CITF. This suggests that protamine’s platelet-inhibitory effects may be more pronounced under conditions approximating arterial blood flow and that its interference with platelet-collagen interaction at high shear is more than its inhibitory effect on platelet aggregation by collagen in a nonflow environment.

View larger version (21K): [in this window] [in a new window]   Figure 3. The effect of heparin 4 and 20 µg/mL, heparin:protamine ratios of 1:1 (40 µg/mL [4 U/mL] heparin and 40 µg/mL protamine), 1:1.5 (40 µg/mL heparin and 60 µg/mL protamine), and 1:2 (40 µg/mL heparin and 80 µg/mL protamine), protamine 4, 20, and 40 µg/mL, on the two Clot Signature Analyzer (CSATM) variables and the activated clotting time (ACT). A, the collagen-induced thrombus formation time (CITF). B, The platelet-mediated hemostasis time (PHT). C, The HepconTM ACT. All samples were compared with simultaneous phosphate-buffered saline controls and are expressed as percentage of paired controls, mean ± SEM, n = 12. *P < 0.05.

View larger version (17K): [in this window] [in a new window]   Figure 4. A, The effect of 100 nM polyarginine, a specific polycationic protein kinase C activator, and 100 nM polylysine, a polycationic negative control, on the two Clot Signature Analyzer (CSATM) variables. All samples were compared with simultaneous phosphate-buffered saline (PBS) controls and are expressed as percentage of paired controls, mean ± SEM, n = 10. *P < 0.05. PHT = platelet hemostasis time; CITF = collagen-induced thrombus formation. B, The effect of the direct thrombin inhibitor, D-phen-ylalanyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK) at final concentrations of 0.1, 1, and 10 µM on the two CSA variables and on the activated clotting time (ACT). All samples were compared with simultaneous PBS controls and expressed as percentage of paired controls, mean ± SEM, n = 10. *P < 0.0001, **P < 0.003.

The PHT tests the platelets’ ability to be activated by and to form an effective hemostatic plug in the presence of extremely high shear (>10,000/s). The PHT was significantly prolonged by protamine alone at concentrations of 20 and 40 µg/mL compared with PBS controls (P < 0.005) (Fig. 3B), but not at a protamine concentration of 4 µg/mL. Interestingly, and unlike the CITF, addition of PL or PA to whole blood did not significantly prolong the PHT compared with PBS controls (Fig. 4A), suggesting that the inhibition of shear-induced platelet activation by protamine is not caused by its highly cationic nature or by PKC activation.

The PHT demonstrated greater sensitivity to the antiplatelet effects of heparin alone than the CITF. Heparin alone at concentrations as small as 0.4 U/mL prolonged the PHT more than threefold compared with PBS controls (P < 0.005) (Fig. 3B). The PHT remained significantly prolonged at a heparin/protamine ratio of 1:1 (P < 0.005) (Fig. 3B). By contrast, a heparin/protamine ratio of 1:1.5 shortened the PHT such that it was not different from PBS controls. As with the CITF, a heparin/protamine reversal ratio of 1:2, producing a 40 µg/mL excess of protamine, significantly prolonged the PHT to 200% of PBS controls (P < 0.05) (Fig. 3B).

Both direct platelet adhesion or activation and thrombin generation resulting in platelet activation may be critical to the platelet hemostatic events that occur at arterial shear, as measured by the CSA; both are inhibited by heparin. Therefore, to explore the relative importance of these possible mechanisms, we used a pure thrombin antagonist, PPACK, devoid of direct platelet-inhibiting action, to explore the sensitivity of the PHT and CITF to thrombin inhibition alone. PPACK at 1.0 µM has equivalent antithrombin activity to heparin at 4 U/mL (40 µg/mL) (22). PPACK coincubation at 1 and 10 µM, but not at 0.1 µM, significantly prolonged the PHT (Fig. 4B). PPACK did not significantly prolong the CITF at any concentration (Fig. 4B). These data suggest that high shear-induced platelet thrombus formation is dependent on both primary platelet functional alterations and secondary thrombin activity on platelets; heparin’s inhibitory effects at high shear, at concentrations as small as 0.4 U/mL, likely result from the inhibition of both physiologies. By contrast, the CITF more uniquely assesses direct platelet function in response to collagen at moderate shear and is less dependent on secondary thrombin activation of platelets.

Given the sensitivity of the PHT to detect thrombin-dependent platelet inhibition, we similarly decided to examine protamine’s effects on thrombin generation by traditional coagulation studies. The PT and PTT in citrated plasma plus protamine were compared with equivalently PBS-diluted control plasma (Table 1). The largest concentration of protamine (80 µg/mL) prolonged both tests beyond the normal range, whereas protamine 40 µg/mL prolonged the PT but not the PTT (Table 1). Hypothesizing that protamine’s cationic charge might be interfering with the negatively charged phospholipid in the assay, we added excess phospholipid to protamine-incubated samples to test for reversal of the prolonged PT and PTT. The addition of excess phospholipid did reverse the protamine-prolonged PTT (Table 1) but did not reverse PT prolongation.

	Discussion

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Platelet function is particularly critical to normal hemostasis under conditions of arterial flow (14). The CSA closely simulates blood flow in intact vessels because platelet and soluble coagulation activation do not occur in the absence of the "punch," or collagen exposure (18,19). This study demonstrates that, under these conditions, heparin alone at concentrations as small as 4 µg/mL and protamine alone at 20 µg/mL potently inhibit platelet function, suggesting that an excess of either drug might impair platelet-dependent hemostasis in vivo. Indeed, heparin reversed with protamine normalizes platelet function only at a 1:1.5 ratio. These data demonstrate antiplatelet effects of heparin and protamine at smaller concentrations and within a narrower therapeutic window for optimizing platelet function by protamine reversal of heparin than previously documented in studies with ACT (12,23–25), PT, PTT, thrombin time (12,26), and platelet aggregometry (2,12,22). Given the widespread practice of calculating protamine dose on the basis of the total heparin dose or the calculated circulating heparin concentration, it is likely that these ratios are easily attainable in the clinical setting.

Previous studies had demonstrated the antiplatelet effects of protamine alone only at large concentrations; the fact that protamine added to heparin more potently inhibited platelet function was attributed to the heparin/protamine complex (2,3,11,12,23). The heparin/protamine complex may reduce platelet P selectin expression (27), platelet aggregation (12) and adenosine diphosphate-mediated platelet Ca²⁺ influx (28). Previous studies, therefore, concluded that reduced platelet aggregation after protamine reversal was caused by the heparin/protamine complex rather than excess protamine (3,12,25). In our study, however, both protamine alone and equivalent concentrations of protamine in excess of heparin (1:2) have similar antiplatelet effects, suggesting that excess protamine directly contributes to platelet dysfunction.

The effects of protamine on the PT and PTT are consistent with previous reports (3) and imply that protamine is capable of inhibiting thrombin formation (Table 1) (2). Our PTT results suggest that the in vitroanticoagulant effects of large concentrations of protamine may also be secondary to nonspecific charge effects because the PTT is corrected by excess negatively charged phospholipid. Whether such an in vitrophenomenon is important is unknown, because patients with very prolonged PTTs caused by antiphospholipid antibodies do not develop clinical bleeding but, paradoxically, develop thrombosis. Further work is needed to determine whether protamine’s interaction with phospholipid on the activated platelet membrane mediates its antiplatelet effect.

Postulated inhibitory mechanisms for the antiplatelet effect of protamine include blockade of the fibrinogen binding site of GP-IIb/IIIa (3), inhibition of platelet factor IV release, and attenuation of platelet response to thrombin (29), as well as direct inhibition of thrombin (3,30,31). The reduced platelet activation response described by Ereth et al. (29) would support our findings of a prolonged CITF. A recent study by Barstad et al. (32) demonstrated significant inhibition of platelet GP-Ib/vWF interaction by protamine. This is one possible mechanism for the inhibition of shear activation of platelets by protamine that we have demonstrated. These authors also demonstrated that increasing the protamine/heparin ratio to 2 markedly impaired or abolished platelet GP-Ib/vWF activity (32), further consistent with our results. Our finding that both the PKC activator PA and the comparatively non-PKC-activating polycationic control PL equally prolong the CITF suggests that a nonspecific charge effect may mediate protamine’s inhibition of platelet response to collagen at moderate shear, perhaps similar to protamine’s ability to prolong the PTT (Table 2). The lack of effect of both PA and PL on the PHT suggests another mechanism for protamine’s inhibition of shear activation, potentially an antithrombin effect, given the sensitivity of the PHT to PPACK (Table 2).

The PHT is prolonged by blockade of GP-Ib or IIb/IIIa, suggesting that both shear-induced platelet adhesion and platelet/platelet aggregation are involved in punch hole closure (18,19). The CITF was less dramatically prolonged by heparin or protamine than by the PHT. This may be because of collagen’s strong agonist capability and the provision of a binding surface that stimulates a more cohesive platelet plug at lower shear stress. The CITF is clearly more sensitive to the antiplatelet effects of protamine compared with platelet aggregometry, and the insensitivity of the CITF to thrombin inhibition makes it ideal for evaluating the specific antiplatelet effects of protamine and heparin.

This study highlights the potential limitations of the ACT after heparin reversal by protamine. The relatively modest concentrations of protamine that inhibit platelet function suggest that protamine excess during empiric heparin reversal may contribute to bleeding. Small-dose protamine administration, based on a heparin/protamine titration method, minimizes throm-bocytopenia and platelet -granule secretion and optimizes platelet response to thrombin (38,39). However, clinical studies to date in cardiac surgical patients have not shown a measurable improvement in hemostasis (40). Further clinical studies are needed to determine whether protamine dosing to optimize platelet function for arterial hemostasis will minimize postoperative blood loss.

In summary, our data demonstrate the potent antiplatelet effects of heparin, particularly on shear activation, detectable at very low heparin concentrations and partly mediated via heparin’s antithrombin activity. The heparin/protamine ratio of 1:1.5 is optimal for reversal of heparin-induced platelet dysfunction, and higher heparin/protamine ratios have significant antiplatelet effects secondary to excess protamine. These data suggest a narrower therapeutic range for protamine in the setting of bleeding caused by platelet dysfunction than suggested by previous studies and suggest that the empiric use of protamine for heparin rebound may adversely affect hemostasis.

	Footnotes

 
Presented in part at the annual meetings of the Society of Cardiovascular Anesthesiologists, Seattle, WA, April 28, 1998, and Chicago, IL, April 27, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ray MJ, Hawson GA, Just SJ, et al. Relationship of platelet aggregation to bleeding after cardiopulmonary bypass. Ann Thorac Surg 1994; 57: 981–6.[Abstract]
2. Kresowik TF, Wakefield TW, Fessler RD, Stanley JC. Anticoagulant effects of protamine sulfate in a canine model. J Surg Res 1988; 45: 8–14.[ISI][Medline]
3. Carr MEJ, Carr SL. At high heparin concentrations, protamine concentrations which reverse heparin anticoagulant effects are insufficient to reverse heparin anti-platelet effects. Thromb Res 1994; 75: 617–30.[ISI][Medline]
4. Saba HI, Saba SR, Morelli GA. Effect of heparin on platelet aggregation. Am J Hematol 1984; 17: 295–306.[ISI][Medline]
5. Carr MEJ, Carr SL, Greilich PE. Heparin ablates force development during platelet mediated clot retraction. Thromb Haemost 1996; 75: 674–88.[ISI][Medline]
6. Shanberge JN, Kambayashi J, Nakagawa M. The interaction of platelets with a tritium-labelled heparin. Thromb Res 1976; 9: 595–609.[ISI][Medline]
7. McDonald K, Chao E. Heparin binding to resting and active platelets. Blood 1989; 74: 238–43.[Abstract/Free Full Text]
8. Sobel M, McNeill P, Carlson P, et al. Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo. J Clin Invest 1991; 87: 1878–93.
9. Kestin AS, Valeri CR, Khuri SF, et al. The platelet function defect of cardiopulmonary bypass. Blood 1993; 82: 107–17.[Abstract/Free Full Text]
10. Khuri SF, Valeri CR, Loscalzo J, et al. Heparin causes platelet dysfunction and induces fibrinolysis before cardiopulmonary bypass. Ann Thorac Surg 1995; 60: 1008–114.[Abstract/Free Full Text]
11. Lindblad B, Wakefield TW, Whitehouse WMJ, Stanley JC. The effect of protamine sulfate on platelet function. Scand J Thorac Cardiovasc Surg 1988; 22: 55–9.[ISI][Medline]
12. Ellison N, Edmunds LHJ, Colman RW. Platelet aggregation following heparin and protamine administration. Anesthesiology 1978; 48: 65–8.[ISI][Medline]
13. Leventhal PS, Bertics PJ. Activation of protein kinase C by selective binding of arginine-rich polypeptides. J Biol Chem 1993; 268: 13906–13.[Abstract/Free Full Text]
14. Ruggeri ZM. Mechanisms initiating platelet thrombus formation. Thromb Haemost 1997; 78: 611–6.[ISI][Medline]
15. Gorog P, Ahmed A. Haemostatometer: a new in vitro technique for assessing haemostatic activity of blood. Thromb Res 1984; 34: 341–57.[ISI][Medline]
16. Gorog P, Kovacs IB. Coagulation of flowing native blood: advantages over stagnant (tube) clotting tests. Thromb Res 1991; 64: 611–9.[ISI][Medline]
17. Muga K, Melton L, Gabriel D. A flow dynamic technique used to assess global hemostasis. Blood Coagul Fibrinolysis 1995; 6: 73–8.[ISI][Medline]
18. Li C, Hoffmann TJ, Hsieh PY, et al. Xylum CSA: automated system for assessing hemostasis in simulated vascular flow. Clin Chem 1997; 43: 1788–9.[Free Full Text]
19. Li CK, Hoffmann T, Hsieh P-Y, et al. The Xylum Clot Signature Analyzer^®: a dynamic flow system that simulates vascular injury. Thromb Res 1998; 92: S67–77.[ISI][Medline]
20. Culliford AT, Gitel SN, Starr N, et al. Lack of a correlation between ACT and plasma heparin during cardiopulmonary bypass. Ann Surg 1981; 193: 105–11.[ISI][Medline]
21. Jobes DR, Aitken GL, Shaffer BS. Increased accuracy and precision of heparin and protamine dosing reduces blood loss and transfusion in patients undergoing primary cardiac operations. J Thorac Cardiovasc Surg 1995; 110: 36–45.[Abstract/Free Full Text]
22. Rubens FD, Weitz JI, Brash JL, Kinlough-Rathbone RL. The effect of antithrombin III-independent thrombin inhibitors and heparin on fibrin accretion onto fibrin-coated polyethylene. Thromb Haemost 1993; 69: 130–4.[ISI][Medline]
23. Levy JH, Cormack JG, Morales A. Heparin neutralization by recombinant platelet factor 4 and protamine. Anesth Analg 1995; 81: 35–7.[Abstract]
24. Kestevan PJ, Ahmed A, Aps C, et al. Protamine sulphate and heparin rebound following open-heart surgery. J Cardiovasc Surg 1986; 27: 600–5.[Medline]
25. Mochizuki T, Olsen P, Szlam F, et al. Protamine reversal of heparin affects platelet aggregation and activated clotting time after cardiopulmonary bypass. Anesth Analg 1998; 87: 781–5.[Abstract/Free Full Text]
26. Ellison N, Ominsky AJ, Wollman H. Is protamine a clinically important anticoagulant? A negative answer. Anesthesiology 1971; 35: 621–9.[ISI][Medline]
27. Ammar T, Fisher CF. The effects of heparinase 1 and protamine on platelet reactivity. Anesthesiology 1997; 86: 1382–6.[ISI][Medline]
28. Mills DC. ADP receptors on platelets. Thromb Haemost 1996; 76: 835–56.[ISI][Medline]
29. Ereth M, Klindworth J, Campbell S. Protamine attenuates agonist-induced platelet signaling/adhesion molecule expression. Anesth Analg 1996; 82: S5.
30. Cobel-Geard RJ, Hassouna HI. Interaction of protamine sulfate with thrombin. Am J Hematol 1983; 14: 227–33.[ISI][Medline]
31. Okano K, Saito Y, Matsushima A, Inada Y. Protamine interacts with the D-domains of fibrinogen. Biochim Biophys Acta 1981; 671: 164–7.[Medline]
32. Barstad RM, Stephens RW, Hamers MJ, Sakariassen KS. Protamine sulphate inhibits platelet membrane glycoprotein Ib-von Willebrand factor activity. Thromb Haemost 2000; 83: 334–7.[ISI][Medline]
33. Endenburg SC, Hantgan RR, Lindeboom-Blokzijl L, et al. On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood. Blood 1995; 86: 4158–65.[Abstract/Free Full Text]
34. Perrault C, Ajzenberg N, Legendre P, et al. Modulation by heparin of the interaction of the A1 domain of von Willebrand factor with glycoprotein Ib. Blood 1999; 94: 4186–94.[Abstract/Free Full Text]
35. Fujimura Y, Titani K, Holland L, et al. A heparin-binding domain of human von Willebrand factor. J Biol Chem 1987; 262: 1734–9.[Abstract/Free Full Text]
36. McDonald K, Horne MK, Chao ES. The effect of molecular weight on heparin binding to platelets. Br J Haematol 1990; 74: 306–12.[ISI][Medline]
37. Ofosu FA, Liu L, Freedman J. Control mechanisms in thrombin generation. Semin Thromb Hemost 1996; 22: 303–8.[Medline]
38. Miyashita T, Nakajima T, Hayashi Y, Kuro M. Hemostatic effects of low-dose protamine following cardiopulmonary bypass. Am J Hematol 2000; 64: 112–5.[ISI][Medline]
39. Shigeta O, Kojima H, Hiramatsu Y, et al. Low-dose protamine based on heparin-protamine titration method reduces platelet dysfunction after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1999; 118: 354–60.[Abstract/Free Full Text]
40. Shore-Lesserson L, Reich DL, DePerio M. Heparin and protamine titration do not improve haemostasis in cardiac surgical patients. Can J Anaesth 1998; 45: 10–8.[Abstract/Free Full Text]

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