Maureane Hoffman

Platelet activity of high‐dose factor VIIa is independent of tissue factor

High‐dose recombinant factor VIIa has been successfully used as therapy for haemophiliacs with inhibitors. The mechanism by which high‐dose factor VIIa supports haemostasis is the subject of some controversy. Postulating a mechanism in which activity is dependent on tissue factor at the site of injury explains the localization of activity but not the requirement for high doses. Postulating a mechanism in which factor VIIa acts on available lipid independently of tissue factor explains the requirement for high doses but not the lack of systemic procoagulant activity. We report that factor VIIa bound weakly to activated platelets (Kd ∼ 90 nm). This factor VIIa was functionally active and could initiate thrombin generation in the presence of plasma concentrations of prothrombin, factor X, factor V, antithrombin III and tissue factor pathway inhibitor. The activity was not dependent on tissue factor. The concentration of factor VIIa required for detectable thrombin generation agreed well with the lowest concentration of factor VIIa required for efficacy in patients. High‐dose factor VIIa may function on the activated platelets that form the initial haemostatic plug in haemophilic patients. These observations are in agreement with clinical trials which have shown that high‐dose factor VIIa was haemostatically effective without causing systemic activation of coagulation.

Platelets and Thrombin Generation

This review examines the evidence that platelets play a major role in localizing and controlling the burst of thrombin generation leading to fibrin clot formation. From the first functional description of platelets, it has been recognized that platelets supply factors that support the activation of prothrombin. Studies have demonstrated that on activation, the amount of one specific lipid, phosphatidylserine, is significantly increased on the outer leaflet of platelet membranes. When it was found that phosphatidylserine containing lipid extracts could be substituted for platelets in clotting assays, this suggested the possibility that changes in platelet lipid composition were necessary and sufficient to account for platelet surface thrombin generation. Because a growing body of data suggest that platelet-binding proteins provide much of the specificity for platelet thrombin generation, we review in this report data suggesting that changes in lipid composition are necessary but not sufficient to account for platelet surface regulation of thrombin generation. Also, we review data suggesting that platelets from different individuals differ in their capacity to generate thrombin, whereas platelets from a single subject support thrombin generation in a reproducible manner. Individual differences in platelet thrombin generation might be accounted for by differences in platelet-binding proteins.

The Effect of Temperature and pH on the Activity of Factor VIIa: Implications for the Efficacy of High-Dose Factor VIIa in Hypothermic and Acidotic Patients

BACKGROUND Recombinant coagulation factor VIIa (FVIIa) is approved for treating hemophiliacs with inhibitors. High-dose FVIIa has also been used off-label to manage hemorrhage in trauma and surgical patients, many of whom also develop hypothermia and acidosis. METHODS We examined the activity of FVIIa on phospholipid vesicles in the presence and absence of tissue factor (TF) and on platelets as a function of temperature and pH. RESULTS FVIIa activity on phospholipids and platelets was not reduced at 33 degrees C compared with 37 degrees C. The activity of FVIIa/TF was reduced by 20% at 33 degrees C compared with 37 degrees C. A pH decrease from 7.4 to 7.0 reduced the activity of FVIIa by over 90% and FVIIa/TF by over 60%. CONCLUSION FVIIa should be effective in enhancing hemostasis in hypothermic patients. However, because the activity of FVIIa is so dramatically affected by pH, its efficacy may be reduced in acidotic patients.

Coagulation disorders and hemostasis in liver disease: Pathophysiology and critical assessment of current management

Normal coagulation has classically been conceptualized as a Y‐shaped pathway, with distinct “intrinsic” and “extrinsic” components initiated by factor XII or factor VIIa/tissue factor, respectively, and converging in a “common” pathway at the level of the FXa/FVa (prothrombinase) complex. Until recently, the lack of an established alternative concept of hemostasis has meant that most physicians view the “cascade” as a model of physiology. This view has been reinforced by the fact that screening coagulation tests (APTT, prothrombin time – INR) are often used as though they are generally predictive of clinical bleeding. The shortcomings of this older model of normal coagulation are nowhere more apparent than in its clinical application to the complex coagulation disorders of acute and chronic liver disease. In this condition, the clotting cascade is heavily influenced by numerous currents and counter‐currents resulting in a mixture of pro‐ and anticoagulant forces that are themselves further subject to change with altered physiological stress such as super‐imposed infection or renal failure. This report represents a summary of a recent multidisciplinary symposium held in Charlottesville, VA. We present an overview of the coagulation system in liver disease with emphasis on the limitations of the current clinical paradigm and the need for a critical re‐evaluation of the current tenets governing clinical practice. With the realization that there is often limited or conflicting data, we have attempted to represent diverse opinion and experience from the perspectives of both hepatology and hematology beginning with a brief update on the physiology of normal coagulation. (HEPATOLOGY 2006;44:1039–1046.)

Platelet functions beyond hemostasis

Summary.  Although their central role is in the prevention of bleeding, platelets probably contribute to diverse processes that extend beyond hemostasis and thrombosis. For example, platelets can recruit leukocytes and progenitor cells to sites of vascular injury and inflammation; they release proinflammatory and anti‐inflammatory and angiogenic factors and microparticles into the circulation; and they spur thrombin generation. Data from animal models suggest that these functions may contribute to atherosclerosis, sepsis, hepatitis, vascular restenosis, acute lung injury, and transplant rejection. This article represents an integrated summary of presentations given at the Fourth Annual Platelet Colloquium in January 2009. The process of and factors mediating platelet–platelet and platelet–leukocyte interactions in inflammatory and immune responses are discussed, with the roles of P‐selectin, chemokines and Src family kinases being highlighted. Also discussed are specific disorders characterized by local or systemic platelet activation, including coronary artery restenosis after percutaneous intervention, alloantibody‐mediated transplant rejection, wound healing, and heparin‐induced thrombocytopenia.

Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2.

Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus.

Platelets induce sinusoidal endothelial cell apoptosis upon reperfusion of the cold ischemic rat liver

BACKGROUND & AIMS Sinusoidal endothelial cell (SEC) apoptosis is a central feature of reperfusion injury in liver transplantation. Platelet sequestration occurs after transplantation with possible deleterious effects. We tested the hypothesis that platelets mediate SEC apoptosis. METHODS Livers were perfused after 24 hours of cold preservation in University of Wisconsin solution in an isolated perfused rat liver model. The perfusate contained isolated syngeneic red blood cells and purified platelets. Effects of inhibiting platelet adhesion on SEC apoptosis was tested using sialyl Lewis-X oligosaccharide (sLe(x)), a natural ligand of selectin adhesion molecules. Reperfusion injury was assessed by established markers of injury. Apoptosis was determined by TUNEL and electron microscopy. RESULTS A third of the circulating platelets was rapidly sequestered in the liver after reperfusion. This was associated with increased graft injury. Single platelets were adherent to sinusoidal lining without morphological or dynamic evidence of impairment of microcirculation. TUNEL staining revealed a 6-fold increase in the number of apoptotic SECs at 1 hour of reperfusion. No hepatocyte death or evidence of necrosis was detected up to 3 hours of reperfusion. Addition of sLe(x) inhibited adhesion and significantly reduced SEC apoptosis. CONCLUSIONS Platelets cause SEC apoptosis upon reperfusion of liver grafts. Prevention of adhesion is protective.

Thrombin Activates Factor XI on Activated Platelets in the Absence of Factor XII

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.

Remodeling the Blood Coagulation Cascade

The concept of a coagulation cascade describes the biochemical interactions of the coagulation factors, but has flaws as a model of the hemostatic process in vivo. For example, the model cannot explain why hemophiliacs bleed when they have an intact factor VIIa/tissue factor (“extrinsic”) pathway. Hemostasis requires the formation of an impermeable platelet and fibrin plug at the site of vessel injury, but it also requires that the powerful procoagulant substances activated in this process remain localized to the site of injury. This control of blood coagulation is accomplished by localizing the procoagulant reactions to events on specific cell surfaces to keep coagulation from spreading throughout the vascular system. A consideration of the critical role of cells allows us to construct a model of coagulation that better explains bleeding and thrombosis in vivo. This cell-based model suggests that the “intrinsic” and “extrinsic” pathways are in fact not redundant systems, but operate in parallel on different cell surfaces.

Hypercoagulation and thrombophilia in liver disease

Summary.  A complex balance exists between endogenous procoagulants and the anticoagulant system in liver disease patients. Hypercoagulable events occur in cirrhosis patients despite the well‐known bleeding diathesis of liver disease. These events may be clinically evident, such as in portal vein thrombosis or pulmonary embolism, but these conditions may also be a silent contributor to certain disease states, such as portopulmonary hypertension or parenchymal extinction with liver atrophy as well as thrombosis of extracorporeal circuits in dialysis or liver assist devices. Moreover, liver disease‐related hypercoagulability may contribute to vascular disease in the increasingly common condition of non‐alcoholic fatty liver disease. Despite the incidence of these problems, there are few widely accessible and practical laboratory tests to evaluate the risk of a hypercoagulable event in cirrhosis patients. Furthermore, there is little research on the use of commonly accepted anticoagulants in patients with liver disease. This article is a result of an international symposium on coagulation disorders in liver disease and addresses several areas of specific interest in hypercoagulation in liver disease. Critical areas lacking clinical information are highlighted and future areas of research interest are defined with an aim to foster clinical research in this field.