title: Delayed, reduced or inhibited thrombin production reduces platelet contractile force and results in weaker clot formation.
abstract: Clot retraction is a thrombin-dependent, platelet-mediated contraction of the cellular clot mass. In this study, the effects of delayed, deficient and inhibited thrombin generation on the development of platelet contractile force (PCF) and clot elastic modulus (CEM) were measured. When normal citrated whole blood is clotted by the addition of exogenous thrombin (1 U/ml) and calcium (10 mmol/l), PCF and CEM start to develop within the first minute and begin to level off by 1200 s. If identical samples are clotted with batroxobin (0.21 microg/ml) and calcium (10 mmol/l), both PCF and CEM development are delayed approximately 5 min. After 1200 s of clotting, however, values in the batroxobin system approach those seen with exogenous thrombin. If the added calcium concentration is held constant at 10 mmol/l, increasing the exogenous thrombin concentration from 0 to 0.5 U/ml results in increased PCF and CEM values. Above 0.5 U thrombin, the effect plateaus. At exogenous calcium of 10 mmol/l, increasing batroxobin concentrations (0-0.210 microg/ml) caused a 75% increase in PCF and a 55% increase in CEM. The increase in CEM reached a plateau above 0.05 microg batroxobin/ml. The effects of varying calcium concentrations were evaluated at constant batroxobin (0.21 microg/ml) and thrombin (1 U/ml) concentrations. With thrombin, PCF and CEM increased by > 700% as CaCl2 increased from 0 to 5 mmol/l. Above 5 mmol/l, no additional increases occurred. With batroxobin, PCF did not develop at CaCl2 concentrations < or = 2.5 mmol/l. Above 2.5 mmol/l CaCl2, PCF values increased and at 10 mmol/l CaCl2 were equal to those seen with thrombin. CEM in batroxobin-mediated clots peaked at 10 mmol/l CaCl2 but were 40% less than the values found in thrombin-mediated clots. When the thrombin inhibitor P-PACK was added to the batroxobin system, dose-dependent decreases in PCF and CEM were noted. At 120 micromol/l, P-PACK totally suppressed PCF. PCF in blood from a factor VIII-deficient patient varied significantly when clotted with batroxobin versus thrombin. PCF development in factor VIII-deficient blood was normal with thrombin but is delayed and depressed with batroxobin. PCF values in factor VIII-deficient blood did not reach the thrombin value after 1200 s of clotting, and CEM was significantly less. These results confirm that PCF development is thrombin dependent and that delay or reduction of PCF development results in structurally weaker clots.
authors.affiliation: Special Coagulation Laboratory, Department of Internal Medicine, Medical College of Virginia, Richmond, Virginia, USA. mcarr@hsc.vcu.edu
authors.forename: M E
authors.initials: ME
authors.lastname: Carr
authors.forename: E J
authors.initials: EJ
authors.lastname: Martin
authors.forename: S L
authors.initials: SL
authors.lastname: Carr
mesh_terms: D000328:Adult; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D012103:Batroxobin / Q000008:administration & dosage / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology*; D001792:Blood Platelets / Q000502:physiology*; D002118:Calcium / Q000008:administration & dosage / Q000494:pharmacology; D003018:Clot Retraction; D004305:Dose-Response Relationship, Drug; D004548:Elasticity; D005260:Female; D006467:Hemophilia A / Q000097:blood; D006801:Humans; D008297:Male; D015539:Platelet Activation / Q000187:drug effects; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000494:pharmacology / Q000502:physiology*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D012103:Batroxobin; D013917:Thrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone; D002118:Calcium