The platelet thrombin binding site(s) has been a controversial issue. Vu and coworkers (4-6) have cloned a platelet thrombin receptor which is a member of the 7-transmembrane loop-receptor family. This receptor is cleaved by thrombin between residues R41 and S42, exposing a new amino-terminal peptide, which serves as a tethered ligand whose binding site resides within the first six amino acids. This tethered ligand peptide binds to an undefined site that induces receptor activation (7-9).Prior to the discovery and cloning of the seven-transmembrane loop receptor, several studies had defined platelet glycoprotein lb (GPIb) as an a-thrombin receptor. These studies described the binding of thrombin to GPIb and the inhibition of a-thrombin binding to GPIb by glycocalicin (a proteolytic fragment of GPIba) and monoclonal antibodies directed against . The decrease or absence of high-affinity a-thrombin binding to Bernard-Soulier platelets, which lack GP~ba (17), provided further evidence that GPIb was a thrombin receptor. In experiments using crosslinking of a-thrombin to human platelets, the only identifiable site of binding was GPIb (18,19).The proposed roles of GPIba in thrombin binding include acting as a high-affinity receptor for localizing a-thrombin near the cell surface or serving as a site for excess thrombin binding (20). In this scheme, GPIba acts primarily as a neutralizing site for excess thrombin generation or as an independent modulator of platelet function through an undefined mechanism. We have studied the high-affinity binding of a-thrombin to human platelets and have examined the inhibitory effects of synthetic peptides derived from portions of the sequence of the GPIba chain and from the seventransmembrane loop thrombin receptor in order to assess the site(s) of high-and moderate-affinity thrombin binding to platelets and thrombin-induced platelet aggregation.MATERIALS AND METHODS Platelet Preparation. Blood was obtained by venipuncture using a two-syringe technique. The blood from the second syringe was placed into polypropylene tubes containing 0.1 ml of sodium citrate anticoagulant, final concentration 10.9 pM. Platelet-rich plasma was prepared from whole blood after centrifugation at 750 x g for 3 min at 250C. Then 6-10 ml of platelet-rich plasma was placed on a discontinuous Larcoll (Sigma) gradient, 20%o (3 ml) and 10% (5 ml), and the platelets were separated from the plasma proteins by centrifugation at 2000 x g for 30 min. The platelets were then resuspended in Hepes buffer at pH 7.35. All platelets were used within 2 hr of being obtained.Thrombin Lalin. Human a-thrombin was prepared and radiolabeled as described (21). Two hundred fifty microliters of a-thrombin solution (1 pg/2 units of activity) was treated with 5 Ad oflactoperoxidase beads, 5 td of0.5 mM KI, 0.5 mCi (18.5 MBq) of Na125I, and 1 A4 of 0.015% H202 for 5 min at room temperature. Free 125I was separated from bound 125I on a 10-ml column of Sephadex G-25 fine equilibrated with 0.05 M Tris/0.1 M NaCl at pH 7.35. Fractions were ...

Platelet von Willebrand factor (vWf) was purified from human platelet concentrates. The multimeric structure of the purified platelet vWf was similar to that observed in the initial platelet lysate, and, like the platelet lysate, the purified platelet vWf contained higher molecular weight multimers than plasma vWf. The apparent molecular weight of the reduced platelet vWf subunit was similar to the plasma vWf subunit. The N-terminal amino acid of the purified platelet and plasma vWf was blocked. In concentration dependent binding to botrocetin- or ristocetin-stimulated platelets, 125I-plasma vWf bound with a higher affinity than platelet. The ristocetin cofactor activity per mg of purified plasma vWf was 5-fold greater than the platelet vWf activity. Platelet and plasma vWf bound to collagen with similar affinities; however, platelet vWf bound to thrombin-stimulated platelets and to heparin with a higher affinity than plasma vWf. The differences in the binding affinity(s) of plasma and platelet vWf to platelet GPIb and GPIIb/IIIa and extracellular matrix proteins may reflect different roles for plasma and platelet vWf in the initial stages of haemostasis and thrombosis.

Thrombocytopenia may accompany variant (type IIB) von Willebrand's disease (vWD) and is thought to result from binding of the abnormal von Willebrand factor (vWF) to the patient's platelets with subsequent platelet aggregate formation and clearance. We have studied a patient with type IIB vWD who became thrombocytopenic during two pregnancies. During the third trimester of pregnancy, her platelet counts dropped to 20,000 to 30,000/microL, and an increase in the intermediate-sized vWF multimers was seen on agarose gel electrophoresis. During this time her platelet-rich plasma showed spontaneous platelet aggregation, and her plasma caused spontaneous aggregation of normal washed platelets. Antibody to platelet glycoprotein Ib completely blocked the spontaneous platelet aggregation, while antibody to platelet glycoprotein IIb/IIIa did not block the response at the concentrations used. Inhibitors of platelet function that elevate platelet cyclic AMP also blocked the response, but aspirin had no effect on the spontaneous platelet aggregation. The patient illustrates that the platelet counts in one individual can vary greatly in type IIB vWD and that the thrombocytopenia that occurs can appear under physiologic conditions that stimulate the endogenous production of the patient's abnormal vWF. The mechanisms leading to spontaneous platelet aggregation and thrombocytopenia appear to be similar to those described for other patients with type IIB vWD.

von Willebrand factor interaction with glycoprotein Ib alpha (GPIb alpha) plays a critical role in the initial phase of platelet adhesion at high shear rates, and it may also play a role in platelet thrombus formation in partially occluded arteries. Previous studies have indicated that two peptides, Cys-474--Pro-488 (peptide 153) and Ser-692--Pro-708 (peptide 154), inhibit von Willebrand factor--GPIb alpha interaction. We have expressed a recombinant fragment of von Willebrand factor, Leu-504--Lys-728 [corrected], with a single intrachain disulfide bond linking residues Cys-509--Cys-695 and examined its ability to inhibit von Willebrand factor--GPIb alpha interactions and platelet adhesion at high shear forces. This recombinant fragment, named VCL, inhibits ristocetin-induced, botrocetin-induced, and asialo-von Willebrand factor-induced platelet aggregation and binding to platelets at an IC50 = 0.011-0.260 microM, significantly lower than the IC50 of peptide 153 or 154, IC50 = 86-700 microM. Peptides 153 and 154 did not result in any inhibition of platelet adhesion (IC50 greater than 500 microM). In contrast, VCL inhibited 50% of platelet adhesion at 0.94 microM and at 7.6 microM inhibited greater than 80% of platelet adhesion to human umbilical artery subendothelium at high shear forces. VCL inhibited the contact and spreading of platelets and also caused a marked decrease in thrombus formation. These studies indicate that VCL may be an effective antithrombotic agent in preventing arterial thrombus formation in areas of high shear force.