Role of Platelet Fibrinogen in the Reactions of Platelets to Thrombin
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Role of Platelet Fibrinogen in the Reactions of Platelets to Thrombin Edward E. Morse, … , Dudley P. Jackson, C. Lockard Conley J Clin Invest. 1965;44(5):809-816. https://doi.org/10.1172/JCI105193. Research Article Find the latest version: https://jci.me/105193/pdf Journal of Clinical Investigation Vol. 44, No. 5, 1965 Role of Platelet Fibrinogen in the Reactions of Platelets to Thrombin * EDWARD E. MORSE,t DUDLEY P. JACKSON, AND C. LocKARD CONLEY (From the Department of Medicine, The Johns Hopkins University and Hospital, Baltimore, Md.) Washed human blood platelets contain a clot- platelets (7). Accordingly, additional studies table protein that is similar to, if not identical were undertaken using enzymes known to alter with, plasma fibrinogen (1-4). After incubation plasma fibrinogen to define more precisely the with trypsin under appropriate conditions, plate- role of platelet fibrinogen in the reaction of plate- lets remain morphologically intact but no longer lets to thrombin. contain clottable protein (5). Trypsinized plate- lets, unlike normal platelets, are not aggregated Methods by fresh serum or by a solution of thrombin and Platelet-rich plasma was obtained from human donors calcium chloride. When resuspended in platelet- by plasmapheresis (8) with plastic equipment' or by free plasma or in a buffered solution of fibrinogen centrifugation of venous blood at 50 g for 30 minutes at containing glucose, trypsinized platelets room temperature. Glassware with which platelets had produce contact was coated with silicone.2 One-tenth volume of retraction of clots formed by thrombin; during a 1.5% solution of the disodium salt of EDTA was used as the formation of the clot and its subsequent re- anticoagulant. Platelet-free plasma was obtained by cen- traction, trypsinized platelets aggregate and un- trifugation at 22,000 g for 10 minutes at 40 C. Platelets dergo the usual changes of viscous metamorpho- were sedimented from 20 to 40 ml of platelet-rich plasma sis (5). These observations suggest that fibrino- by centrifugation at 1,900 g for 10 minutes at room tem- perature. The sedimented platelets were resuspended in gen on the surface of platelets is involved in the 5 ml of EDTA Tris-buffered saline (equal volumes 0.3 reaction of platelets to thrombin. This reaction M Tris solution and 0.9% sodium chloride, pH adjusted does not consist simply of the coagulation of fib- to 7.5 with 2 N hydrochloric acid and containing EDTA, rinogen with entrapment of platelets in the fibrin 0.0013 M). The platelets were washed twice by cen- mesh. Under usual conditions divalent cations trifugation at 4,700 g for 5 minutes at 40 C and resus- are required for thrombin-induced platelet aggre- pension in 5 ml of the EDTA Tris-buffered saline. They gation but not for clotting of fibrinogen (5, 6). were then resuspended in EDTA Tris-buffered saline, Electron photomicrographs of platelet aggregates and various amounts of one of the following proteolytic enzymes were added: bovine thrombin,s trypsin,4 plas- show no striated fibrin strands between adherent minogen prepared by the modified Kline method (9) from human plasma Fraction III 5 and activated with * Submitted for publication October 2, 1964; accepted streptokinase,s spontaneously activated plasmin in 50% January 21, 1965. glycerol,7 a-chymotrypsin,8 papain,9 or carboxypepti- These investigations were carried out under contract AT(30-1)1208 between the Atomic Energy Commission and The Johns Hopkins University and were supported 'Fenwal, Framingham, Mass. in part by research grant HE-01601 from the National 2 G.E. silicone SC-87 dri-film. Heart Institute, research career program award AM-K3- 3Thrombin, topical, Parke, Davis, Detroit, Mich. 3779, and graduate training grant T1 AM-5260 from the 4 Twice recrystallized, salt-free trypsin, Worthington National Institute of Arthritis and Metabolic Diseases. Biochemical Corp., Freehold, N. J. Presented in part at the meeting of the Federation of 5 Cohn Fraction III human plasma, Lederle Labora- American Societies for Experimental Biology in April tories, Pearl River, N. Y. 1963, and abstracted in Fed. Proc. 1963, 22, 505. Varidase 2200-76, Lederle Laboratories, Pearl River, t Work done in part during the tenure of U. S. Public N. Y. The activated plasminogen had an activity of 30 Health Service special fellowship CSP 18,037 from casein U per mg. the National Cancer Institute. Address requests for re- T Forty-four caseinolytic U per ml, kindly supplied by prints to Dr. Edward E. Morse, Hematology Division, Dr. Alan Johnson, New York, N. Y. This preparation The Johns Hopkins Hospital, Baltimore, Md. 21205. was dialyzed against 0.15 M KG (pH 7.3) to remove the 809 810 EDWARD E. MORSE, DUDLEY P. JACKSON, AND C. LOCKARD CONLEY dase.10 The platelets were incubated with the enzyme for thrombin, 100 U per ml in 0.025 M CaCl2. The tubes 10 minutes at 370 C in a final volume of 5 ml. During were observed constantly for 6 minutes, then periodically incubation with thrombin an EDTA concentration of for 2 hours and again at 24 hours. 0.008 M was used to inhibit aggregation. In some ex- Serotonin. One ml was centrifuged at 22,000 g for 10 periments inhibitors were added following the incubation minutes, and the platelets were frozen and thawed five to assure cessation of enzyme activity. e-Aminocaproic times in 3.0 ml of 0.02 N hydrochloric acid. The sero- acid (EACA) 11 or a bovine lung inhibitor 12 was used tonin concentration of 2.0 ml of the suspension was de- to inhibit plasmin, soy bean trypsin inhibitor 13 to inhibit termined by a modified spectrophotofluorometric method trypsin, and heparin14 to inhibit thrombin. The plate- (10) and was related to the protein concentration, which lets then were immediately centrifuged in the cold and was measured on 0.5 ml by the method of Sutherland, washed once with and resuspended in 5 ml EDTA Tris- Cori, Haynes, and Olsen (11). Serotonin release was buffered saline. Samples of these suspensions were used expressed as percentage of decrease in the treated plate- for the following studies. lets as compared to controls. Aggregation. Five-tenths ml was centrifuged at 1,800 ATP. In six experiments 3.0-ml portions of suspen- g for 5 minutes, and the platelets were suspended in 0.2 sions of platelets that had been incubated with thrombin, ml Tris-buffered saline without EDTA in siliconized trypsin, or plasmin were centrifuged at 22,000 g for 10 tubes to which was added 0.05 ml of a solution of throm- minutes and the platelets lysed in 2.0 ml of cold 3% tri- bin, 100 U per ml in 0.025 M CaCl2. The tubes were chloroacetic acid. The sediment was removed by cen- gently agitated and observed for gross and microscopic trifugation at 22,000 g for 10 minutes and the supernatant aggregation. Aggregation was graded as follows: 0, no chromatographed on a Dowex 1 anion exchange column aggregation; 1 +, few small clumps; 2 +, numerous small that was 4 cm long and 1.0 cm in diameter. Two ml of clumps; 3 +, many large clumps; 4 +, very large clumps distilled water was added to the column after addition of with few free platelets. the 2.0 ml of supernatant. The acid-soluble nucleotides Clot retraction. Five-tenths ml was centrifuged at were isolated by intermittent gradient elution with 4.0-ml 1,800 g for 5 minutes, and the platelets were resuspended volumes of formic acid and mixtures of formic acid and in 0.3 ml platelet-free plasma in siliconized tubes to ammonium formate as described by Siekevitz and Potter which was added 0.1 ml of a solution of thrombin, 100 (12). The procedure was modified to include elution U per ml in 0.025 M CaCl2. The tubes were inverted with mixtures of 4.0 N formic acid with 1.0 M and 4.0 M once and incubated at 370 C. Clot retraction was graded ammonium formate. The eluates were read at 260 my at 2 hours and again at 24 hours: 0, no retraction; 1 +, with a Beckman model DU spectrophotometer. separation of clot from wall of tube; 2 +, clot retracted The activity of the streptokinase-activated plasminogen to half the volume in the tube; 3 +, clot retracted to one- was measured by the method of Norman (13). The pro- quarter of the volume in the tube; and 4+, small re- teolytic (caseinolytic) activities of some of the enzymes tracted clot similar to control. In experiments with plas- were compared using a pH stat (14). min-treated platelets, 0.025 ml of EACA was added to the plasma before the addition of thrombin and calcium. Clottable protein. Three ml was centrifuged at 22,000 Results g for 10 minutes, and the platelets were frozen and thawed five times in 0.2 ml of a 0.9% solution of sodium chloride Washed human blood platelets incubated with with alcohol-dry ice. The suspension was centrifuged at various concentrations of thrombin in the presence 22,000 g for 10 minutes and the supernatant tested for of EDTA did not aggregate or lyse during the clottable protein by the addition of 0.1 ml of a solution of incubation (Table I). Serotonin was released glycerol and was concentrated to the original volume by from the platelets by concentrations of thrombin negative pressure. that did not completely inactivate clottable pro- 8 a-Chymotrypsin 3 X crystallized, Worthington Bio- tein. Higher concentrations of thrombin inacti- chemical Corp., Freehold, N. J. vated the clottable protein, and such platelets no 9 Papain, twice crystallized suspension in 0.05 M sodium acetate, Worthington Biochemical Corp., Freehold, N.