Mechanical recording of reptilase-clot retraction = effect of adenosine-5′-diphosphate and prostaglandin E1

Greilich

1996

Thromb Haemost

SummaryAlthough snake venom enzymes such as reptilase do not cause viscous metamorphosis, platelet secretion or clot retraction; when batroxobin and calcium are added to citrated blood significant platelet force development occurs. When this batroxobin-calcium system was applied to the study of platelet function during cardiopulmonary bypass (CPB), force development was found to be completely inhibited. After heparin reversal by protamine sulfate, significant recovery of force occurred. The present investigation was performed to evaluate the role of heparin in reducing force development during CPB. At concentrations above 0.10 U/ml, heparin totally suppressed force development in normal plasma. Addition of protamine sulfate to heparinized plasma caused complete recovery of force development. These concentrations of heparin had little effect on platelet aggregation by ADP or collagen. Possible direct effects of heparin on fibrin assembly and structure were studied by adding varying amounts of heparin to plasma and then inducing clot formation with batroxobin. At 1 U/ml, heparin reduced the size of fibrin fibers by 33%. Higher heparin concentrations had no additional effect. These results indicate that heparin may be responsible for a significant component of the decreased platelet force noted during cardiopulmonary bypass. To test whether heparin’s effect could be due to suppression of thrombin activity, the effects of the antithrombin hirudin on force development were measured. Hirudin also inhibited force development in a concentration dependent manner. Thus, heparin’s reduction of platelet force development may be due, at least in part, to suppression of thrombin activity.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Heparin Ablates Force Development during Platelet Mediated Clot Retraction

Greilich

1996

Thromb Haemost

“…Since batroxobin has already produced a fibrin network, PCF can be measured from its onset and serves as a marker of thrombin generation. Similar reptilase clot retraction assays have proven useful in evaluating effects of agents on platelet function (20,21).…”

Section: Utility Of the Batroxobin Based Pcf Assay As A Measure Of Thmentioning

confidence: 99%

Effects of recombinant factor VIIa on platelet function and clot structure in blood with deficient prothrombin conversion

Martin²,

Kuhn³

et al. 2003

Thromb Haemost

SummaryWhile recombinant factor VIIa (rFVIIa) shows promise as a broad-spectrum hemostatic agent, questions remain regarding the most appropriate dose and the best way to monitor its effects. In this study we tested the sensitivity of a thrombin dependent platelet assay, platelet contractile force, to the effects of rFVIIa in normal, factor-deficient, and inhibitor-containing blood samples. Dose dependent effects of rFVIIa on platelet contractile force (PCF) and clot elastic modulus (CEM) were measured in all blood samples.rFVIIa minimally affected PCF and CEM in normal blood clotted with thrombin or batroxobin. While rFVIIa minimally altered PCF and CEM in factor VIII (FVIII) deficient blood clotted with thrombin, rFVIIa increased PCF and CEM and shortened the lag phase in a dose dependent manner in batroxobin-induced clots. The effects of rFVIIa in factor IX (FIX) deficient blood mirrored the effects seen in FVIII deficient samples. Whether clotted with thrombin or batroxobin, baseline PCF and CEM were abnormally low in FVIII deficient samples containing FVIII inhibitors. In such samples, rFVIIa caused dose dependent improvement of PCF, CEM, and lag phases. In one patient with a spontaneous inhibitor, rFVIIa caused dose dependent increases in PCF and CEM in blood clotted with either enzyme.rFVIIa corrects the deficient thrombin generation seen in FVIII and FIX deficiency, and in blood containing FVIII inhibitors. As a consequence, platelet function is improved and clot structure is enhanced. Platelet contractile force and clot elastic modulus measurements are sensitive to the dose dependent effects of rFVIIa.

“…Since thrombin generation must be followed by fibrin clot formation before PCF can begin to occur in the recalcification assay, the lag phase tends to be longer and more variable. Similar assays termed reptilase clot retractions were shown in the early 1970s to be sensitive to a variety of antiplatelet and anticoagulant agents [13].…”

Section: Methodsmentioning

confidence: 99%

Onset of force development as a marker of thrombin generation in whole blood: the thrombin generation time (TGT)

Journal of Thrombosis and Haemostasis

Martin

Kuhn

et al. 2003

Summary. Prothrombin activation requires the direct interplay of activated platelets and plasma clotting factors. Once formed, thrombin causes profound, irreversible activation of platelets and reinforces the platelet plug via ®brin formation. Delayed or de®cient thrombin production increases bleeding risk. Commonly employed coagulation assays, the prothrombin and partial thromboplastin times, use clot formation as a surrogate marker of thrombin generation. These assays routinely utilize platelet-poor plasma and completely miss the effects of platelets. Other markers of thrombin generation, prothrombin fragment 1 2 (F1 2) and thrombin±antithrombin complex, are typically measured after the fact. We report a simple assay, which employs the onset of platelet contractile force (PCF) as a surrogate marker of thrombin generation. PCF generation occurs concomitant with the burst of F1 2 release. The time between assay start and PCF onset is termed the thrombin generation time (TGT). TGT is prolonged in clotting factor de®ciencies and in the presence of direct and indirect thrombin inhibitors. TGT shortens to normal with clotting factor replacement and shortens with administration of recombinant factor VIIa. TGT is short in thrombophilic states such as coronary artery disease, diabetes and thromboangiitis obliterans and prolongs toward normal with oral and intravenous anticoagulants.