Evolution of blood coagulation and fibrinolysis

The key steps in the evolution of blood coagulation and fibrinolysis have been reconstructed from an analysis of the molecular evolution of their constituents. The data suggest that the blood coagulation and complement cascades are descendants of an ancestral defence system that served the dual role of immobilization and destruction of invading bacteria and the prevention of loss of body fluids. The enzymes of the fibrinolytic, tissue-remodelling cascades form a distinct group, more closely related to the proteases of the digestive tract than to the components of the blood coagulation and complement cascades. Molecular evolution of these enzymes therefore suggests that they are descendants of an ancestral protease responsible for degradation of extracellular proteins. It is shown that the regulatory extensions of the proteases of the blood coagulation, fibrinolytic and complement cascades were assembled from domains borrowed from other proteins. Most non-protease components of these systems were also constructed by this evolutionary mechanism.     

Snake venom prothrombin activators homologous to blood coagulation factor Xa

We have recently determined the complete amino acid sequence of trocarin, a group D prothrombin activator from the venom of Tropidechis carinatus (Australian rough-scaled snake). This proteinase is both functionally and structurally similar to mammalian blood coagulation factor Xa. It shows approximately 70% homology and possesses the characteristic Gla domain, two EGF domains and serine proteinase domain. To examine structure-function relationships, we generated a molecular model of trocarin based on a human factor Xa des-Gla crystal structure (1xka) as template. Based on known sites of interaction between mammalian factor Xa, factor Va and prothrombin, structure-function relationships of trocarin were explored. Unlike factor Xa, trocarin is glycosylated and has a large carbohydrate moiety at the entrance to its active site pocket. This might contribute to differences observed in the kinetics of hydrolysis of synthetic substrates by trocarin as compared to human factor Xa. A Ca(2+)-binding loop present in the heavy chain of factor Xa also seems to be lost in trocarin. In addition to its role in hemostasis, factor Xa shows other biological effects, including inflammation via its interaction with effector protease receptor-1 (EPR-1). Interestingly, the EPR-1 recognition site is distinctly different in trocarin, the functional consequences of which are being investigated.     

Development of the human coagulation system in the healthy premature infant

This study was designed to determine the postnatal development of the human coagulation system in the healthy premature infant. Consecutive mothers of healthy premature infants born at either St Joseph's Hospital or McMaster University Medical Centre in Hamilton were asked for consent. One hundred thirty-seven premature infants (30 to 36 weeks of gestational age) entered the study. The premature infants did not have any major health problems and did not require ventilation or supplemental oxygen. Demographic information and a 20-mL blood sample were obtained in the postnatal period on days 1, 5, 30, 90, and 180. Between 40 and 96 premature infants were studied on each day for each of the following tests: prothrombin time, activated partial thromboplastin time, thrombin clotting time, plasminogen; 13 factor assays [fibrinogen, II, V, VII, VIII, IX, X, XI, XII, XIII, high-mol-wt kininogen (HMWK), prekallikrein (PK), von Willebrand factor (vWF)] and eight inhibitors [antithrombin III (AT-III), heparin cofactor II, alpha 2-antiplasmin, alpha 2-macroglobulin, alpha 1-antitrypsin, C1 esterase inhibitor, protein C (PC), and protein S (PS)]. A combination of biologic and immunologic assays were used. Between 30 to 36 weeks there was a minimal effect of gestational age for levels of AT-III, PC, and factors II and X only; therefore, the entire data set was used to generate reference ranges for these components of the coagulation system for premature infants. Next, the results for the premature infants were compared with those of a previously published study in 118 fullterm infants and with those for adults. An effect of gestational age was shown for plasminogen, fibrinogen, factors II, V, VIII, IX, XI, XII, HMWK, and all eight inhibitors. In general, the postnatal maturation towards adult levels was accelerated in premature infants as compared with the fullterm infants. By 6 months of age, most components of the coagulation system in premature infants had achieved near adult values.     

Factors and inhibitors of blood coagulation and fibrinolysis in acute nonlymphoblastic leukaemia

Factors of coagulation and fibrinolysis have been evaluated in 15 patients with untreated acute nonlymphoblastic leukaemia (ANLL). 10 patients had major bleeding (MB) and 6 had laboratory signs of DIC. 5 patients went into complete remission (CR). Antithrombin III (AT III) was decreased in 7 patients, antiplasmin (AP) in 9, fibronectin (FN) in 6 and factor XIII in 4/12. The ratio between factor VIIIR:Ag and factor VIII:C was over 2.0 in 11 patients, and high values were especially seen in patients with MB and patients with DIC. Spontaneous proteolytic activity, measured with S-2288 was increased in 3 patients who all had MB, and none of whom achieved CR. 2 patients with promyelocytic leukaemia (M3) had low fibrinogen and AP, high FDP and normal AT III, speaking for primary fibrinolysis, which in addition to proteolytic enzymes in the blast cells are important contributing factors regarding MB in ANLL.     

Allele-specific activators and inhibitors for kinesin

Members of the kinesin superfamily are force-generating ATPases that drive movement and influence cytoskeleton organization in cells. Often, more than one kinesin is implicated in a cellular process, and many kinesins are proposed to have overlapping functions. By using conventional kinesin as a model system, we have developed an approach to activate or inhibit a specific kinesin allele in the presence of other similar motor proteins. Modified ATP analogs are described that do not activate either conventional kinesin or another superfamily member, Eg5. However, a kinesin allele with Arg-14 in its nucleotide binding pocket mutated to alanine can use a subset of these nucleotide analogs to drive microtubule gliding. Cyclopentyl-ATP is one such analog. Cyclopentyl-adenylylimidodiphosphate, a nonhydrolyzable form of this analog, inhibits the mutant allele in microtubule-gliding assays, but not wild-type kinesin or Eg5. We anticipate that the incorporation of kinesin mutants and allele-specific activators and inhibitors in in vitro assays should clarify the role of individual motor proteins in complex cellular processes.     

Circulating inhibitors of blood coagulation associated with procainamide-induced lupus erythematosus

We studied a patient being treated with procainamide in whom we observed a high antinuclear antibody titer and prolonged activated partial thromboplastin (PTT), prothrombin (PT), and Stypven times (ST). Serum antibody concentrations against single-stranded DNA were elevated while those against native DNA were not elevated, suggesting the procainamide-induced lupus syndrome. Dilution of the patient's plasma with normal plasma failed to correct the PTT and PT, indicating the presence of an inhibitor(s) to blood coagulation. The anticoagulant activity was associated with the IgG fraction of the patient's serum. Addition of purified or partially purified human factors IX, X, VIII, VII, XIa, prekallikrein, high molecular weight kininogen, or phospholipids to the patient's plasma failed to correct the PTT, PT, or ST; however, purified human factor XII and prothrombin corrected the PTT and ST, respectively. These results indicate that production of antibodies directed against antigenic determinants on coagulation proteins can be a manifestation of procainamide-induced lupus erythematosus.     

Natural inhibitors of blood coagulation and fibrinolysis in patients with lupus anticoagulant.

We studied the natural inhibitors (NI) of blood coagulation and fibrinolysis in 50 patients with lupus anticoagulant (LA), in order to identify possible alterations of these NI, that could favour thrombotic manifestations. We found no statistically significant difference in antithrombin III, protein C and alpha 2-antiplasmin between controls and patients with LA, irrespective of their clinical manifestations. We found an increase of plasminogen activator inhibitor (PAI, P < 0.001) and a decrease of free protein S (PSf, P < 0.001) and total protein S (PSt, 0.01 < P < 0.05) in the patients with LA when compared with the control group. We found no difference in the levels of NI between patients with thrombosis (n = 19) and without thrombosis (n = 31) nor between patients with (n = 25) or without thrombosis and/or foetal loss (n = 25). In contrast, we observed a decrease of PSf in women with foetal loss (n = 10) as compared with women without foetal loss (n = 22, 0.01 < P < 0.05) and a decrease of PSf when comparing 19 patients with systemic lupus erythematosus (SLE) with 31 patients without SLE (0.01 < P < 0.05). These findings show that the patients with LA had several abnormalities in the NI system, but there was no significant association between levels of PAI, PSf, PSt and a history of thrombosis.     

An alternative extrinsic pathway of human blood coagulation

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.     

Pharmacokinetic allometric scaling of coagulation factors and tissue-type plasminogen activators

Summary.  The objective of this study was to evaluate the predictive performance of pharmacokinetic interspecies scaling of coagulation factors and tissue-type plasminogen activators to predict clearance, volume of distribution at steady-state and half-life in humans from animal data. The human pharmacokinetic parameters were predicted using one, two or at least three animal species. The results of the study indicated that the pharmacokinetic parameters of coagulation factors and tissue-type plasminogen activators can be predicted with reasonable accuracy in humans when three or more animal species are available. Two-species scaling can also be performed, but the accuracy of predicted parameters is less than three-species scaling.     

Plasminogen activators and their inhibitors in leukemic cell homogenates

Plasminogen activator (PA) and PA inhibitor (PAI) were measured in homogenates of leukemia cells. Both PA and PAI levels were higher in non-lymphoblastic leukemia than in lymphoblastic leukemia. The levels were below the sensitivity of determination in chronic myelocytic leukemia (CML) but showed significant increases in blast crisis (CML, bc). The level of the tissue type PA (t-PA) antigen was highest in acute myelobtestic leukemia (AML) and that of the urokinase type PA (u-PA) was highest in acute promyeiocytic leukemia (APL). The PAH antigen showed no marked cell specificity, but the PAI-II antigen was markedly increased in myelomonocytic leukemia and acute monocytic leukemia (AMoL). From these findings, various PAs and PAIs are considered to be present in leukemia cells and to be involved in hemostatic disorders, thus they are of diagnostic value in leukemia. © 1993 Wiley-Liss, Inc.