Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6086-6090, October 1983 Medical Sciences Inhibition of thromboxane A2 synthesis in human platelets by coagulation factor Xa (arachidonic acid/factor V antibody/thrombin) A. KUMAR SINHA, A. KONETI RAO, JANET WILLIS, AND ROBERT W. COLMAN Thrombosis Research Center and Hematology-Oncology Section of the Department of Medicine, Temple University School of Medicine, Philadelphia, PA 19140 Communicated by Oscar D. Ratnoff, June 23, 1983 ABSTRACT Factor X. binds to platelets provided that factor with platelets that leads to the efficient formation of thrombin Va is present on the platelet surface, an interaction that results in from prothrombin, a concomitant effect of this binding of factor a striking acceleration of the conversion of prothrombin to throm- Xa is the inhibition of TXA2 synthesis in these cells. Because bin. Thrombin then initiates fibrin formation, induces platelet ag- TXA2 is a platelet-aggregating agent and a potent vasoconstric- gregation, and stimulates the intraplatelet synthesis of throm- tor, the inhibition of TXA2 synthesis by factor Xa would provide boxane A2 (TXA2). Addition of thrombin (2.4-14.4 nM) to platelet- negative feedback control of the synthesis of TXA2 in platelets. rich plasma increased the basal level of TXA2, measured as thromboxane B2, from <0.5 pmol per 108 platelets to (mean + MATERIALS AND METHODS SEM) 100 ± 22 and 250 ± 10 pmol per 108 platelets, respectively. Treatment of platelet-rich plasma with increasing concentrations Chemicals and Reagents. [1-`4C]Arachidonic acid (55.8 mCi/ of factor X. (1-12 nM) prior to the addition of thrombin pro- mmol; 1 Ci = 3.7 x 1010 Bq) and [5,6,8,9,11,12,14,15-3H(N)] gressively inhibited the production of TXA2. Thrombin (9.6 nM), thromboxane B2 (TXB2) (100 Ci/mmol) were purchased from which produced 93% of the maximal formation of TXA2, was in- New England Nuclear. [5,6,8,11,12,14,15-3H(N)]Prostaglan- hibited 70% by factor Xa (10 nM). To identify which of these steps ,din E2 (PGE2) (160 Ci/mmol) was bought from Amersham. Di- in thromboxane synthesis was inhibited by factor Xa, platelets la- isopropyl fluorophosphate (iPr2P-F) was bought from Sigma. beled with [14C]arachidonic acid were exposed to thrombin and Antisera to PGE2 was obtained from Miles. All other chemicals products of prostaglandin synthesis were separated by thin-layer used were of analytical grades. chromatography. In contrast to the inhibition of TXA2 synthesis, Coagulation Factors. Bovine factor X was prepared by the prostaglandin E2 and prostaglandin F2a synthesis were not inhib- method of Bajaj and Mann (5). The purified factor X was ac- ited suggesting that neither phospholipase(s) nor cycloxygenase was tivated by using the Russell's viper venom and the resulting involved. The inhibition of TXA2 formation by factor Xa could be factor Xa purified according to Jesty and Esnouf (6). Polyacryl- reversed by increasing the molar ratio of thrombin to factor Xa amide gel (8.6%) electrophoresis of factor Xa in the presence to 5.5. Incubation of platelets with an IgG fraction of a human of dithiothreitol showed the presence of three bands corre- monoclonal antifactor V antibody, previously shown to inhibit fac- sponding to heavy chain of aXa (Mr = 34,500), heavy chain of tor X. binding, was found to block factor Xa inhibition of TXA2 synthesis. The inhibition of TXA2 synthesis requires the presence (3Xa (Mr = 29,500), and light chain of I3Xa (Mr = 16,000) (7), of the active site serine of factor Xa and is not specific for TXA2 the major component accounting for 95%. The prepared factor formation induced by thrombin because it is also demonstrable Xa had a specific activity of 5,500 units per mg of protein de- when the agonist is ADP. Further, factor Xa does not require ad- termined according to Cole et al. (8). The molarity of factor Xa ditional plasma components for its action because its inhibitory was obtained by adding the molecular weights of heavy chain effects are detected in gel-filtered platelets. The effect of factor of PXa and the light chain (Mr = 45,000). Xa was evident at physiological (1.3 mM) calcium concentrations. Both bovine factors X and Xa were also obtained from Sigma. These results indicate that factor X. binding to platelets through Factor X was found to be a single component (Mr = 54,000) on factor Va not only stimulates thrombin formation but also has a 8.6% polyacrylamide gel electrophoresis in NaDodSO4 (0.1%) countervailing effect by inhibiting TXA2 formation. but in the absence of reducing agent. The specific activity of factor X was 100-150 units/mg of protein. Factor Xa used had The interaction of blood coagulation factor Xa with the human a specific activity of 4,000-5,000 units/mg of protein. Poly- platelet surface in the presence of Ca2" accelerates the gen- acrylamide gel electrophoresis of factor Xa in the presence of eration of thrombin from prothrombin at a rate 300,000 times NaDodSO4 and dithiothreitol showed the presence ofthree bands faster than the rate observed with factor Xa in solution (1). Fac- corresponding to the heavy chain of aXa (Mr = 34,500, 10%), tor Xa binding to platelets requires the presence of factor Va, heavy chain of /3Xa (Mr = 29,500, 90%), and light chain (Mr = which may be derived by release from platelets (1) from its in- 16,000). Except that the commercial factor Xa was somewhat tracellular site, the a granules (2), by agonists such as thrombin. less active than the proteinase prepared as described above, the Alternatively, exogenous factor Va produced by the action of enzyme was essentially identical in both preparations. thrombin on plasma factor V (3) facilitates factor Xa binding in Human thrombin was purified by the method of Fenton et a coordinate manner. In addition to these two actions of throm- al. (9) by using commercial thrombin as the starting material bin on platelets and factor V, respectively, thrombin is also a from Sigma. The purified material was a homogenous protein potent inducer of thromboxane A2 (TXA2) synthesis in these in gel electrophoresis and had a specific activity of 2,480 units/ cells (4). mg. The molarity of the thrombin preparation was calculated We now report that in addition to the interaction of factor Xa from the molecular weight of the enzyme, Mr 33,500. The publication costs of this article were defrayed in part by page charge Abbreviations: TXA2, thromboxane A2; TXB2, thromboxane B2; PGF2a, payment. This article must therefore be hereby marked "advertise- prostaglandin F2a; PGE2, prostaglandin E2; iPr2P-F, diisopropyl fluo- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. rophosphate; RIA, radioimmunoassay. 6086 Downloaded by guest on September 26, 2021 Medical Sciences: Sinha et al. Proc. Natl. Acad. Sci. USA 80 (1983) 6087 iPr2P-F-inactivated factor Xa was prepared by incubating 1.0 Antifactor V Antibody. Antifactor V antibody used in these mg of factor Xa, prepared as above, in 25 mM Tris'HCl buffer studies was a IgG fraction of a human monoclonal IgG4 A an- (pH 8.0) with 15 mM iPr2P-F in a total volume of 0.5 ml for 2.5 tifactor V antibody (19) kindly supplied by Helen Glueck (Uni- hr at 370C as described by Leveson and Esnouf (10). Treatment versity of Cincinnati). An IgG fraction prepared by (NH4)2SO4 of factor Xa by iPr2P-F does not result in total loss of activity, precipitation and DEAE-Sephadex chromatography of the an- but under these conditions we found that factor Xa lost >85% tibody was prepared and furnished by H. C. Chiu (Thrombosis of its original activity. The reaction mixture was then dialyzed Research Center). Approximately 40 Mg of the IgG fraction per against 0.15 M NaCl for 16 hr at 40C. ml neutralized >99% offactorV activity in 1 ml of normal plasma. Human factor X was prepared according to DiScipio et al. (11), was activated and purified as described above (6), and had RESULTS a specific activity of 4,000 units/mg of protein. Stimulation of TXA2 Formation by Thrombin. Addition of Platelet Preparation. All blood donors had not taken any increasing concentrations of thrombin to platelet-rich plasma medication at least 2 wk before blood donation. Venous blood induced the synthesis of higher levels of platelet TXA2, as de- was collected through siliconized needles into plastic syringes; termined by RIA of TXB2 (Fig. 1). Although as little as 0.2 unit coagulation was prevented by mixing 9 vol of blood with 1 vol of thrombin per ml (2.4 nM) would aggregate 80% of the plate- of trisodium citrate (final concentration, 13 mM). Platelet-rich lets (not shown), the formation of TXB2 continued to increase, plasma was obtained by centrifugation of samples for 10 min at up to a thrombin concentration of 14.4 nM. The basal level of 170 x g. The supernatant platelet-rich plasma (2-3 X 10' cells TXB2 (<0.5 pmol per 108 platelets) increased -300-fold (100 per ml) was collected. Platelet counts were performed in a ± 22 pmol per 108 cells; mean ± SEM) and 800-fold (250 ± 10 Coulter Counter model ZB equipped with a 50-Mum aperture pmol per 108 cells) in the presence of 2.4 nM and 14.4 nM tube. Gel-filtered platelets were prepared as described (12). thrombin, respectively. The labeling of gel-filtered platelets with [I4C]serotonin and its Inhibition of TXB2 Production by Factor Xa. When platelet- release were studied according to Walsh et al. (13). rich plasma was treated with 9.6 nM thrombin, the TXA2 level Platelet Aggregation Studies.