During the past several years, we have initiated studies to determine the role of plasma factors and platelets, and the properties of the blood vessel, which influence the activation of the coagulation mechanism on the subendothelium. Studies were performed by exposing everted segments of de-endothelialized rabbit aorta, mounted in a perfusion chamber, to non-anticoagulated human blood for 5 to 10 minutes under a range of flow conditions, and measuring fibrin and platelet deposition on the subendothelium, and fibrinopepstide A (FPA) levels in post-chamber blood. In normal subjects, platelet deposition increased progressively with increasing shear rates (50-2600 sec-1 ), whereas fibrin deposition and FPA levels decreased sharply at shear rates greater than 650 sec-1 . To examine the role of plasma coagulation factors, we utilized a shear rate of 650 sec-1 to study patients with severe deficiencies of factors XII, XI, IX or VIII. In contrast to the partial thromboplastin time (PTT), which was most strikingly abnormal in patients with factor XII or XI deficiency, fibrin deposition and FPA levels were greater in patients deficient in factor XII or XI than in those with factor VIII or IX deficiency. In addition, we observed smaller platelet thrombi in hemophilia (but not afibrinogenemia), suggesting that thrombin influenced the formation of platelet thrombi under these shear conditions. The findings suggested that tissue factor-Vila activation of factor IX could be important in mediating fibrin deposition on subendothelium and might explain why patients with factor XII deficiency (and some with factor XI deficiency) do not bleed. Initial studies to demonstrate tissue factor activity in subendothelium were inconclusive. More recently, utilizing shorter (1.5, 2 and 3 min) perfusion periods, we have observed decreased fibrin deposition and FPA levels in patients with factor VII deficiency and we have obtained further support for the presence of tissue factor in subendothelium in experiments utilizing a monoclonal antibody to tissue factor. Our studies suggest that activation of factor IX by tissue factor-Vila could account for the results obtained in patients with plasma coagulation defects. Direct experimental verification of this hypothesis will require more extensive studies on the kinetics governing the activation of coagulatjon factors on the subendothelium. In subsequent studies, we examined the role of platelets in mediating fibrin deposition. At a shear rate of 650 sec-1 we found (utilizing patients with thrombocytopenia) that platelets were required for fibrin deposition ; little or no fibrin was deposited on the subendothelium when platelet adhesion was less than 4%, corresponding to blood platelet counts less than 5000/ul. Studies performed in patients with functional platelet disorders provided additional information on the specific platelet properties that contribute to fibrin deposition at this shear rate. Decreased fibrin deposition was observed in a patient with Scott Syndrome, a disorder characterized by an impaired capacity of the platelets to catalyze the conversion of factor X to factor Xa (in the presence of factor IXa and VIII) and prothrombin to thrombin (in the presence of factor Va), the latter defect owing to a decreased factor Xa-binding capacity of the platelets. In contrast to the findings in Scott Syndrome, both fibrin deposition and FPA values were completely normal (and possibly increased) in patients with glycoprotein Ilb/IIIa deficiency. In patients with glycoprotein lb deficiency, the major defect was an impaired association of fibrin with platelets, but not subendothelium. The findings in patients with functional platelet disorders indicate that a monolayer of platelets (including those deficient in glycoprotein Ilb/IIIa) is completely active in promoting fibrin deposition on subendothelium. In addition, they suggest that an agent capable of inducing a platelet defect similar to that observed in Scott Syndrome might prevent platelet-fibrin thrombi at shear rates (200-800 sec-1 ) comparable to those in the coronary circulation. Studies performed at a variety of shear rates in both normal subject^ and patients with platelet disorders suggested that, under the conditions used, platelets were essential for fibrin formation at intermediate (650 sec-1 ), but not low (50 sec-1 ) shear rates. Since platelets have been shown to bind activated coagulation proteins (such as factor Xa, Va, and IXa) to their surface, the presence of adherent platelets on the subendothelium could, with increasing shear rates, serve to maintain activated coagulation proteins in the .boundary layer at a concentration that would otherwise be reduced through convective diffusion in their absence. Thus, at low shear rates (50 sec-1 ), the concentration of activated coagulation factors in the boundary layer might be sufficient to support fibrin deposition despite the absence of platelets, whereas at very high shear rates (2,600 sec-1 and above), even the presence of platelets is insufficient to maintain the required concentration. The shear-dependent defect of fibrin formation that we observed in Scott Syndrome is consistent with such a theory. The results of our various studies demonstrate the complex role of blood flow, plasma coagulation factors, specific platelet properties, and the procoagulant properties (tissue factor) of the vessel in mediating subendothelium-induced coagulation and suggest further experiments for studying the mechanisms involved.
GPVI Interaction with Polymerized Fibrin Boosts Thrombin Generation and Thrombus Growth