A Thrombin-Sensitive Protein of Human Platelet Membranes

Abstract

The action of thrombin on intact human platelets has been studied with the aid of polyacrylamide gel electrophoresis in sodium dodecylsulfate. A single major membrane protein band with a molecular weight of 190,000 disappears after thrombin treatment, while a new membrane protein with a molecular weight of 107,000 appears. This may represent hydrolysis of the thrombinsensitive protein. When platelets are disrupted or when the thrombin-sensitive protein is solubilized from membranes prior to thrombin treatment, no hydrolysis occurs. The effect of thrombin on the platelet membrane protein is complete within 2 min which suggests that hydrolysis of this membrane protein may trigger the physiological effects of thrombin on platelets.Platelets are anucleate circulating particles, with a diameter of 2-4 Mum in man, which function in hemostasis. These "cells" are metabolically active and catalyze the formation of fatty acids, phospholipids, glycogen, and even protein (1). Both glycolytic and Krebs cycle oxidative pathways are active in these cells.Blood platelets are rapidly converted from free floating cells to an aggregated mass of intact platelets during the process of hemostasis or of thrombus formation. This obvious change in the platelet surface is accompanied by a variety of morphological and biochemical changes which have been termed "viscous metamorphosis" (1). The most potent agent known to induce this process is the proteolytic enzyme thrombin, which is generated from reactions of the coagulation pathway. Characterization of the role of thrombin in producing viscous metamorphosis is integral to understanding thrombus formation and may also provide some insight into the membrane specification of cell-cell interactions.Previous work done in our laboratory (2) has shown that thrombin causes a 7-fold increase in the rate of incorporation of glycerol into phosphatidyl serine in the platelet membrane. This burst in the rate of lipid synthesis is maximal within 2 min of the addition of thrombin and such synthesis returns to the pre-thrombin rate by 20 min. Studies of the mechanism of this thrombin effect indicate that proteolysis is involved, since the effect can be mimicked by trypsin and inhibited by prior incubation of trypsin with soybean trypsin inhibitor. Human platelets, collected and isolated as described previously (2), were washed twice in a pH 6.5 isotonic buffer containing 0.113 M NaCl, 0.0043 M K2HPO4, 0.0043 M\I Na2HPO4, 0.0244 M NaH2PO4, and 1 mg/ml glucose. After washing, l)latlets were resuspended in either 0.154 M NaCl-0.154 A Tris HCl (pH 7.4) 9:1 with 1 mg/ml glucose, or in a pH 7.5 buffer containing 0.109 M NaCl, 0.043 M K2HP04, 0.0106 M Na2HPO4, 0.0083 MI NaH2PO4, and 1 mg/ml glucose.Platelets were incubated at 370C at 1-2 X 109 cells/ml in resuspension buffer with or without 1 U/ml thrombin and 2.5 mM CaCl2. The cells were disrupted by no-clearance-pestle homogenization (5), glycerol loading, and hypotonic lysis (6) Samples were solubilized in 1-3%o SDS-1%o 0-mercaptoethanol -0.1 AM so...

Isolation and Characterization of Myosin and Two Myosin Fragments from Human Blood Platelets

“…were mixed with two volumes of 0.6 MI KCl-15 mMN Tris -HCl (pH 7.5, hereafter referred to as Buffer A), 10 mMI sodium pyrol)hosphate, 5 mMI M9gCl2, and 10 mMI DTT; the cells were lysed by making the solution 3% in n-butyl alcohol. After extraction for 16 hr, the lysate was centrifuged for 1 hr at 27,000 X g, yielding the extract supernatant (see Table 1). Thrombosthenin was )recipitated from this supernatant by dilution with 2 volumes of 2 mM MgSO4 and adjustment of the pH to 6.4 (4).…”

Cartilage oligomeric matrix protein and thrombospondin 1

“…Thrombospondin 1 (TSP1) is another non-collagenous matrix glycoprotein that was originally identified as a platelet granule protein (Baenziger et al, 1971). Endogenous TSP has been identified by immunological methods in a number of tissues (Frazier, 1987(Frazier, , 1991Wight et al, 1985) and among these in articular cartilage and the meniscus McDevitt, 1988, 1991).…”

Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer

“…Thrombospondin (TSP) is a high-molecular-weight multifunctional glycoprotein which was first described as a product of platelets (Baenziger et al, 1972), released from the alpha granules in response to activation of thrombin (Lawler et al, 1986). TSP is not only one of the components of the extracellular matrix (ECM) in a wide variety of tissues (O'Shea et al, 1988;O'Shea et al, 1990), but also contains binding sites for a number of ECM and cell-associated molecules (Taraboletti et al, 1987;Sage et al, 1991).…”