Purification and characterization of recombinant antistasin: a leech-derived inhibitor of coagulation factor Xa

Antistasin (ATS) is a selective, tight-binding inhibitor of blood coagulation Factor Xa originally isolated from the salivary glands of the Mexican leech Haementeria officinalis. In order to provide sufficient quantities of ATS to further investigate the role of Factor Xa in blood coagulation, a recombinant version of ATS has been produced in an insect baculovirus host-vector system. In this study, we describe the purification and in vitro and in vivo characterization of a single recombinant antistasin (rATS) isoform. The purified protein constitutes a minor isoform relative to the more abundant ATS isoforms present in leech salivary gland extracts. In vitro, rATS inhibits purified human Factor Xa stoichiometrically, prolongs plasma-based clotting assays at nanomolar concentrations, and like native ATS, is cleaved at a single position by Factor Xa during the course of inhibition. An initial evaluation of the in vivo efficacy of rATS was addressed utilizing a rhesus monkey model of mild disseminated intravascular coagulation. rATS was shown to fully suppress thromboplastin-induced fibrinopeptide A generation in a dose-dependent fashion. The availability of rATS should provide a valuable tool for the critical evaluation of the specific role played by Factor Xa in coagulation.     

Evaluation of the efficacy and safety of rivaroxaban using a computer model for blood coagulation

Rivaroxaban is an oral, direct Factor Xa inhibitor approved in the European Union and several other countries for the prevention of venous thromboembolism in adult patients undergoing elective hip or knee replacement surgery and is in advanced clinical development for the treatment of thromboembolic disorders. Its mechanism of action is antithrombin independent and differs from that of other anticoagulants, such as warfarin (a vitamin K antagonist), enoxaparin (an indirect thrombin/Factor Xa inhibitor) and dabigatran (a direct thrombin inhibitor). A blood coagulation computer model has been developed, based on several published models and preclinical and clinical data. Unlike previous models, the current model takes into account both the intrinsic and extrinsic pathways of the coagulation cascade, and possesses some unique features, including a blood flow component and a portfolio of drug action mechanisms. This study aimed to use the model to compare the mechanism of action of rivaroxaban with that of warfarin, and to evaluate the efficacy and safety of different rivaroxaban doses with other anticoagulants included in the model. Rather than reproducing known standard clinical measurements, such as the prothrombin time and activated partial thromboplastin time clotting tests, the anticoagulant benchmarking was based on a simulation of physiologically plausible clotting scenarios. Compared with warfarin, rivaroxaban showed a favourable sensitivity for tissue factor concentration inducing clotting, and a steep concentration-effect relationship, rapidly flattening towards higher inhibitor concentrations, both suggesting a broad therapeutic window. The predicted dosing window is highly accordant with the final dose recommendation based upon extensive clinical studies.     

Activation of coagulation releases endothelial cell mitogens

Recent studies have indicated that endothelial cell function includes elaboration of growth factors and regulation of coagulation. In this paper we demonstrate that activated coagulation Factor X (Factor Xa), a product of the coagulation mechanism generated before thrombin, induces enhanced release of endothelial cell mitogens, linking these two functions. Mitogenic activity generated by cultured bovine aortic endothelial cells in response to Factor Xa included platelet-derived growth-factor-like molecules based on a radioreceptor assay. Effective induction of mitogens by Factor Xa required the integrity of the enzyme's active center and the presence of the gamma-carboxyglutamic acid-containing domain of the molecule. Factor Xa-induced release of mitogens from endothelium occurred in serum-free medium and was not altered by hirudin or antibody to Factor V, indicating that it was a direct effect of Factor Xa and was not mediated by thrombin. Elaboration of mitogenic activity required only brief contact between Factor Xa and endothelium, and occurred in a time-dependent manner. Generation of enhanced mitogenic activity in response to Factor Xa was unaffected by the presence of actinomycin D and was not associated with increased hybridization of RNA from treated cells to a v-sis probe. Release of mitogenic activity was dependent on the dose of Factor Xa, being half-maximal at 0.5 nM and reaching a maximum by 5 nM. Radioligand binding studies demonstrated a class of endothelial cell sites half-maximally occupied at a Factor Xa concentration of 0.8 nM. The close correspondence between the parameters of Factor Xa-induced mitogen release and Factor Xa binding suggests these sites may be related. When Factor X was activated on the endothelial cell surface by Factors IXa and VIII, the Factor Xa formed resulted in the induction of enhanced release of mitogenic activity. These data suggest a mechanism by which the coagulation system can locally regulate endothelial cell function and vessel wall biology before thrombin-induced release of growth factors from platelets.     

Studies, with a luminogenic peptide substrate, on blood coagulation Factor X/Xa produced by mouse peritoneal macrophages

The formation and secretion of coagulation Factor X/Xa by mouse peritoneal macrophages was studied with a luminogenic peptide substrate (S-2613; t-butyloxycarbonylisoleucylglutamyl-γ-piperidylglycylarginylisoluminol). Amidolysis was quantified by measuring the light emitted during oxidation of isoluminol, released by Factor Xa. A lower detection limit of about 0.5ng of Factor Xa was established; the assay was linear with enzyme concentration up to at least 100ng/ml. Factor X was determined after treatment with the Factor X-activating component of Russell's-viper (Vipera russelli) venom. Macrophages, cultured in the absence of serum, released Factor X/Xa into the culture medium. The concentration of coagulation enzyme in the medium increased in an essentially linear fashion over a period of at least 3 days, at a rate corresponding to 6–8ng produced/24h per 10⁶ cells. The ratio of Factor Xa/X+Xa varied from about 60 to 100%, showing that activation of Factor X to Xa is not prerequisite to release of the enzyme from the cells. Factor Xa activity was suppressed in the presence of warfarin [3-(α-acetonylbenzyl)-4-hydroxycoumarin; 12.5μg/ml of medium], but could be restored by adding vitamin K (0.1μg/ml) along with the warfarin. Cultures to which Sepharose beads containing covalently bound anti-(Factor X) antibodies had been added showed decreased amounts of free Factor X/Xa in the culture medium. The missing activity could be demonstrated by incubating the recovered conjugate with the substrate peptide S-2613. Factor Xa produced by the macrophages was efficiently inactivated by heparin in the presence of antithrombin, heparin with high affinity for antithrombin being more effective than the corresponding low-affinity species.     

Preparation and properties of derivatives of bovine factor X and factor Xa from which the gamma-carboxyglutamic acid containing domain has been removed

Limited proteolysis of bovine blood coagulation Factor X by chymotrypsin produces a derivative in which the light chain is cleaved between Tyr 44 and Lys 45. Two peptide products, residues 1-44 of the Factor X light chain and a modified zymogen, Factor X(-GD) have been isolated and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution behavior on anion-exchange chromatography, amino acid composition, and by partial amino acid sequence determination. Factor X(-GD) no longer contains the 12 gamma-carboxyglutamic acid residues of the native zymogen and thus serves as a model for investigation of the properties conferred on Factor X by the presence of gamma-carboxyglutamic acid. Cleavage of Factor X at Tyr 44 by chymotrypsin is inhibited by Ca2+ and Mg2+ ions. Factor X(-GD) is activated by the coagulation factor activator of Vipera russellii venom, but at less than 1% of the rate of activation of native Factor X. The susceptibility of Tyr 44 to chymotryptic cleavage implies that this residue is on the surface of the light chain of Factor X. Factor Xa(-GD) is indistinguishable from native Factor Xa in its activity on Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, on prothrombin alone, and on prothrombin plus Factor Va. In the presence of phospholipid the rate of prothrombin activation catalyzed by Factor Xa(-GD) is the same as in the absence of phospholipid.     

Proteolytic alterations of factor Va bound to platelets

The coagulation protein Factor Va forms the receptor for the serine protease Factor Xa at the platelet surface. This membrane-bound complex of Factor Va and Factor Xa plus calcium constitutes the enzymatic complex prothrombinase, which effects the conversion of prothrombin to the clotting enzyme, thrombin. Studies were undertaken to investigate the proteolytic events accompanying the inactivation of platelet-bound Factor Va by activated protein C as well as the ability of Factor Xa to protect Factor Va from activated protein C inactivation. During the course of these studies, observations were made which indicated that Factor Va was also cleaved by both a platelet-associated protease, as well as Factor Xa. When Factor Va was incubated with washed platelets, electrophoresis and autoradiography of solubilized platelet pellets indicated that three Factor Va peptides were associated with the platelet: component D (Mr = 94,000), component E (Mr = 74,000), and a 90,000-dalton peptide (component D') which appeared with time as the result of a platelet-associated protease cleavage of component D. The Factor Va peptides bound to platelets were proteolytically inactivated by activated protein C, resulting in five peptide products, all of which remained associated with the platelet-membrane surface. Factor Va was protected from activated protein C proteolysis by complex formation with Factor Xa or active site-blocked Factor Xa. However, active Factor Xa cleaved platelet-bound Factor Va to peptide products which also remained associated with the platelet. Whereas activated protein C rapidly cleaved components D and D' with secondary cleavages occurring in component E, Factor Xa rapidly cleaved component E with secondary cleavages occurring in components D and D'. The Factor Xa-cleaved Factor Va is catalytically functional. To determine whether cleavage was necessary for function, prothrombin conversion reaction mixtures were monitored for thrombin formation and Factor Va cleavage with time in a defined phospholipid vesicle model system. The results indicated that Factor Xa cleavage of Factor Va is not essential for Factor Va activity but may promote its ability to function in the prothrombinase complex.     

In vivo bypass of hemophilia A coagulation defect by factor XIIa implant

Hemophilia A and B coagulation defects, which are caused by deficiencies of Factor VIII and Factor IX, respectively, can be bypassed by administration of recombinant Factor VIIa. However, the short half-life of recombinant Factor VIIa in vivo negates its routine clinical use. We report here an in vivo method for the continuous generation of Factor VIIa. The method depends on the implantation of a porous chamber that contains Factor Xa or XIIa, and continuously generates Factor VIIa bypass activity from the subject's own Factor VII, which enters the chamber by diffusion. Once inside, the Factor VII is cleaved to Factor VIIa by the immobilized Factor Xa or XIIa. The newly created Factor VIIa diffuses out of the chamber and back into the circulation, where it can bypass the deficient Factors VIII or IX, and enable coagulation to occur. In vitro, this method generates sufficient Factor VIIa to substantially correct Factor VIII-deficient plasma when assessed by the classical aPTT coagulation assay. In vivo, a Factor XIIa peritoneal implant generates bypass activity for up to one month when tested in rhesus monkeys. Implantation of such a chamber in a patient with hemophilia A or B could eventually provide a viable alternative to replacement therapies using exogenous coagulation factors.     

The design of phenylglycine containing benzamidine carboxamides as potent and selective inhibitors of factor Xa

Factor Xa, a critical serine protease in the blood coagulation cascade, has become a target for inhibition as a strategy for the invention of novel anti-thrombotic agents. Here we describe the development of phenylglycine containing benzamidine carboxamides as novel, potent and selective inhibitors of factor Xa.     

Activation of coagulation factor V by calcium-dependent proteinase

Factor V is a key coagulation cofactor, regulating the rate of Factor Xa-catalyzed prothrombin conversion. Activation of Factor V markedly accelerates coagulation. This study describes a new class of Factor V activators, sulfhydryl proteinases. Of the enzymes studied, calcium-dependent proteinase was the most effective activator. Activation of Factor V by this enzyme was associated with cleavage of 125I-labeled Factor V to peptides distinct from those generated by previously described activators. Calcium-dependent proteinase-activated Factor Va peptides with molecular weights of 114,000 and 93,000 bound both to Factor Xa and to cultured endothelial cells. Calcium-dependent proteinase was identified in vascular endothelial cells, a tissue that also synthesizes Factor V. These findings suggest a previously unknown mechanism for cellular regulation of coagulation.     

The activation of coagulation factor X. Identity of cleavage sites in the alternative activation pathways and characterization of the COOH-terminal peptide.

Bovine Factor X can be activated by two alternative pathways. The first, favored at high concentrations of the complex of tissue factor and Factor VII, is initiated by the action of Factor VII on Factor X to cleave an activation peptide from the NH2 terminus of the heavy chain, to produce alpha-Xa. This is then converted autocatalytically to another form of Factor Xa, beta-Xa, by the loss of a 17-residue glycopeptide from the COOH terminus of the heavy chain, in a lipid-dependent reaction. The alternative pathway, favored at lower activator concentrations, is initiated by the action of Factor Xa on Factor X, in the presence of lipid, to release the same COOH-terminal peptide as is produced in the conversion of alpha-Xa to beta-Xa. The intermediate produced by the loss of this peptide from Factor X,I1, can be activated directly to beta-Xa by the tissue factor-Factor VII complex, with the loss of the same NH2-terminal peptide as is produced in the conversion of Factor X to alpha-Xa. The autocatalytic activation of Factor X by Factor Xa described previously occurs to a marked extent only at very low activator concentrations, and has been shown to proceed largely by the loss of the normal NH2-terminal peptide from the heavy chain of I1-Initial experiments show that neither peptide affects the rate of coagulation by either the extrinsic or intrinsic pathways. The amino acid sequences have been determined on both sides of the peptide cleavages, and it has been shown that the cleavage sites are the same, regardless of the pathway of activation. The amino acid sequence and carbohydrate composition of the COOH-terminal peptide have been determined. The carbohydrate moiety is attached via an O-glycosidic linkage at a threonine residue, and contains galactosamine but no glucosamine.     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.     

Human prothrombinase complex assembly and function on isolated peripheral blood cell populations

A membrane-bound Ca2+-dependent complex of the cofactor Factor Va and the enzyme Factor Xa comprises the prothrombinase coagulation complex which catalyzes the proteolytic conversion of prothrombin to thrombin. Analyses of the kinetics of prothrombin activation permit calculation of the stoichiometry and binding parameters governing the functional interactions of Factor Va and Factor Xa with isolated thrombin-activated human platelets and isolated leukocyte subpopulations. Our kinetic approach indicates that Factor Xa binds to approximately 2700 +/- 1000 (n = 8) functional sites on the surface of thrombin-activated platelets with an apparent dissociation constant (Kd) equal to 1.18 +/- 0.53 X 10(-10) M and kcat equal to 19 +/- 7 mol of thrombin/s/mol of Factor Xa bound. The store of Factor V in normal platelets prevents an analogous determination of the functional Factor Va platelet binding sites. Factor Va and Factor Xa titrations performed using platelets from a Factor V antigen-deficient individual indicate that Factor Va and Factor Xa form a 1:1 stoichiometric complex on the surface of thrombin-activated platelets. Both binding isotherms are governed by the same apparent Kd (approximately equal to 10(-10) M) and expressed the same kcat/site (14-17 s-1. Factor Xa-platelet binding parameters are not altered by the use of different platelet agonists, the choice of anticoagulant, or platelet washing procedure. Kinetics of prothrombin activation indicate also that monocytes, lymphocytes, and neutrophils possess, respectively, 16,000, 45,000, and 8,000 Factor Va-Factor Xa receptor sites/cell, which are all governed by apparent KdS approximately equal to 10(-10) M. Enzymatic complexes bound to monocytes or neutrophils exhibit kcat values similar to the platelet-bound complex. Complexes bound to lymphocytes are only 25% as active.     

Homology modeling and structural validation of tissue factor pathway inhibitor

Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either “Tissue Dependent Pathway” (also known as extrinsic pathway) or by “contact activation pathway” (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex and Factor Xa. TFPI is one of the most studied coagulation pathway inhibitor which has various clinical and potential therapeutic applications, however, its exact mechanism of inhibition is still unknown. Structure based mechanism elucidation is commonly employed technique in such cases. Therefore, in the current study the generated a complete TFPI structural model so as to understand the mechanistic details of it''s functioning. The model was checked for stereochemical quality by PROCHECK-NMR, WHATIF, ProSA, and QMEAN servers. The model was selected, energy minimized and simulated for 1.5ns. The result of the study may be a guiding point for further investigations on TFPI and its role in coagulation mechanism.     

Sulfonamidolactam inhibitors of coagulation factor Xa

As part of an effort to identify novel backups for previously reported pyrazole-based coagulation Factor Xa inhibitors, the pyrazole 5-carboxamide moiety was replaced by 3-(sulfonylamino)-2-piperidone. This led to the identification of a structurally diverse chemotype that was further optimized to incorporate neutral or weakly basic aryl and heteroaryl P1 groups while maintaining good potency versus Factor Xa. Substitution at the sulfonamide nitrogen provided further improvements in potency and as did introduction of alternate P4 moieties.     

Unfractionated heparin inhibits thrombin-catalysed amplification reactions of coagulation more efficiently than those catalysed by factor Xa.

We have proposed previously that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent amplification reactions, and that prothrombinase is formed in heparinized plasma only after Factor Xa activates Factor VIII and Factor V. These propositions were based on the demonstration that both heparin and Phe-Pro-Arg-CH2Cl completely inhibited 125I-prothrombin activation for up to 60 s when contact-activated plasma (CAP) was replenished with Ca2+. Furthermore, the addition of thrombin to CAP before heparin or Phe-Pro-Arg-CH2Cl completely reversed their inhibitory effects. Additional support for the above hypotheses is provided in this study by demonstrating that, when the activity of thrombin is suppressed by heparin (indirectly) or by Phe-Pro-Arg-CH2Cl (directly), exogenous Factor Xa reverses the ability of these two agents to inhibit prothrombin activation. Prothrombin activation was initiated by adding Factor Xa (1 nM) or thrombin (1 or 10 nM) simultaneously with CaCl2 to CAP. In the absence of heparin or Phe-Pro-Arg-CH2Cl, prothrombin activation was seen 15 s later in either case. Heparin failed to delay, and Phe-Pro-Arg-CH2Cl delayed for 15 s, prothrombin activation in CAP supplemented with Factor Xa. In contrast, heparin and Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation for at least 45 s in CAP supplemented with 1 nM-thrombin. Heparin failed to delay prothrombin activation in CAP supplemented with 10 nM-thrombin, whereas Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation in this plasma for 45 s. These results suggest that in CAP: (1) Factor Xa can effectively activate Factor VIII and Factor V when the proteolytic activity of thrombin is suppressed; (2) heparin-antithrombin III is less able to inhibit Factor Xa than thrombin; (3) suppression of the thrombin-dependent amplification reactions is the primary anticoagulant effect of heparin.     

超声刀联合双极电凝在开放甲状腺手术中的临床应用

目的:探讨超声刀联合双极电凝在开放式甲状腺手术中的临床应用价值。方法:选取我科2015年1月至2016年12月收治的224例甲状腺肿瘤患者,随机分为2组,A组112例患者采用超声刀和双极电凝进行手术,B组112例采用传统丝线结扎方法,2组均为开放式甲状腺手术。比较两组患者的手术时间、术中出血量、术后引流管拔出时间、住院时间以及术后并发症发生率。结果:A组手术时间、术中出血量、术后引流管拔出时间、住院时间分别为(58.7±10.5)min、(17.1±7.1)m L、(2.8±0.3)d、(8.8±0.5)d,均低于B组的(78.6±11.7)min、(41.5±8.2)m L、(3.9±0.3)d、(11.1±0.6)d,差异有统计学意义(P0.05);A组术后总并发症发生率为4.46%(5/112),低于B组的11.61%(13/112),差异有统计学意义(P0.05)。结论:在开放式甲状腺手术中使用超声刀联合双极电凝,可明显提高手术效果,降低术后并发症的发生率,从而提升甲状腺手术的安全性。     

胸腔镜辅助小切口与传统开胸手术对胸外伤患者凝血功能及预后的影响

目的:探讨胸腔镜辅助小切口(VTAMS)与传统开胸手术对胸外伤患者凝血功能及预后的影响。方法:回顾性选取2017年7月~2019年2月期间我院收治的胸外伤患者91例,根据手术方式的不同分为A组(n=44,行传统开胸手术)和B组(n=47,行VTAMS手术),比较两组患者围术期指标、术前及术后7d的凝血功能指标、术后并发症发生情况及病死率。结果:B组术中出血量、术中输血量少于A组,手术时间、住院时间短于A组(P<0.05)。两组患者术后7d活化部分凝血酶原时间(APTT)、血小板(PLT)较术前降低,D-二聚体(DD)、凝血酶原时间(PT)较术前升高(P<0.05);B组术后7d的APTT、PLT高于A组,DD、PT则低于A组(P<0.05)。B组术后并发症发生率为10.64%(5/47),低于A组的27.27%(12/44)(P<0.05)。B组病死率为2.13%(1/47),低于A组的15.91%(7/44)(P<0.05)。结论:与传统开胸手术相比,胸外伤患者应用VTAMS手术治疗,可改善围术期指标,对凝血功能影响较轻,减少术后并发症的同时还可降低病死率。     

A prothrombinase complex of mouse peritoneal macrophages

Addition of prothrombin to mouse peritoneal macrophages in vitro resulted in the formation of a thrombin-like enzyme, as demonstrated by use of the luminogenic peptide substrate S-2621. The prothrombinase activity was sedimented by high-speed centrifugation following homogenization of the cells and was abolished by treatment of the cells with the nonionic detergent Triton X-100 at 0.02% concentration. Moreover, the activity was drastically reduced by maintaining cultures in the presence of warfarin and, presumably due to competitive substrate inhibition, by adding S-2222, a chromogenic peptide substrate for Factor Xa. These findings suggest that prothrombin cleavage is catalyzed by Factor Xa at the macrophage surface. The generated thrombin was inhibited by antithrombin, and this reaction was accelerated by heparin with high affinity for antithrombin but not by the corresponding oligosaccharides composed of 8-14 monosaccharide units. Such oligosaccharides which are capable of accelerating the inactivation of Factor Xa by antithrombin, inhibited thrombin formation from prothrombin in the macrophage cultures, presumably by promoting inactivation by antithrombin of Factor Xa in a prothrombinase complex. Activation of the macrophage coagulation system, as proposed to occur in certain inflammatory conditions, thus may be modulated at various levels by heparin, or heparin oligosaccharides, released from mast cells.     

A novel approach in potential anticoagulants from peptides epitope 558–565 of A2 subunit of factor VIII

Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser⁵⁵⁸–Gln⁵⁶⁵ region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa–FIXa interaction. Based on this, a series of linear peptides, analogs of the 558–565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.     

Anatomical and medical laboratory parallels of various forms of thrombus formation

77 patients who had suffered from various diseases and had died under clinical circumstances of thromboembolic complications were systematically examined in clinical and morphological respect. Depending on the pathological-anatomical character of thrombus formation determined they were classified into 4 groups: 1. Massive venous thromboses with embolus of the lung; 2. Disseminated intravasal coagulation; 3. Locally limited, mainly arterial thromboses or some microthromboses; 4. Without any thrombotic complications. A distinct dependence between the changes in the standard values of the coagulation status (fibrinogen, thrombocytes, prothrombin time, bleeding and coagulation time) and the character and extent of thrombotic processes could not be detected. It is solely the level of fibrin splitting products which, to a certain degree, reflects the extent and spread of thrombotic processes.