Stable Fibrin Clot Research Articles

A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates

Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa—TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa—TF complex, but also in the interaction of FVII with both its activators and cofactor TF.

Protein crosslinking, tissue transglutaminase, alternative splicing and neurodegeneration

Increasing interest and awareness of protein aggregation as being implicated in neurodegenerative processes has developed in recent years. One novel mechanism for this may be transglutaminase (TGase)-mediated protein crosslinking, that is involved in a variety of natural processes ranging from the stabilization of fibrin clots to production of the epidermal cell envelope and the fluid barrier of the skin. TGases are also implicated in both function and dysfunction of the central (CNS) and peripheral (PNS) nervous systems. The most ubiquitously expressed member of the TGase family, known as tissue TGase (tTG) or TG2, which, in addition to catalyzing the production of ε-lysine to γ-glutaminyl isodipeptide bonds, serves a dual function as the G-protein Gα h and is both expressed and active in PNS and CNS. It differs from other members of the TGase gene family in this regard and may implicate it in ‘switches’ from life or trophic signaling to those associated with apoptosis. In this regard, recent data indicate that one or more TGases are involved in neurodegenerative disorders such as the Qn/CAG repeat disorders, as well as Alzheimer's and Parkinson's diseases. As do many genes, particularly those highly expressed in the CNS, tTG undergoes alternative processing. Elevated expression and alternative splicing, resulting in a short (S) isoform of tTG with more active crosslinking activity, are associated with increased neuronal loss in affected regions in the demented brain. Our recent and novel data indicate that tTG mRNA, protein, and TGase activity are elevated in certain neurodegenerative diseases, and are accompanied by transcription of this S splice variant that results in unregulated crosslinking, unique to neurodegenerative disorders.

Interleukin 6 and Haemostasis

Interleukin 6 and Haemostasis

The role of fibrin sealants in hemostasis

Hemostasis is a prerequisite for wound healing, and under normal physiologic conditions, it is achieved by means of the coagulation cascade. However, there are a number of surgical procedures where there may be considerable benefits to the patient, surgeon, or health-care costs if hemostasis can be achieved more efficiently. The rapid and effective control of bleeding during and after surgery reduces blood loss and can help reduce postoperative complications. These improved outcomes can reduce the need for transfusion, with the associated risk of viral transmission, and have a positive impact on operative and hospital stay times. Fibrin sealants are surgical hemostatic agents derived from human plasma that reproduce the final steps in the coagulation pathway and form a stable fibrin clot. Fibrin sealants are used in a broad range of surgical procedures to assist hemostasis, including cardiovascular, hepatic, and splenic surgery, gastrointestinal hemorrhage, skin grafting, and dental extractions in anticoagulated patients. Patients with coagulopathies are at high risk of prolonged or excessive bleeding during or after invasive surgery, and these patients may also benefit from the use of fibrin sealants. This article reviews the role of fibrin sealants in hemostasis, citing a number of key clinical studies that report a significant reduction in blood loss or chest drain output after surgery with fibrin sealant compared with controls.

Recombinant factor XIII improves established experimental colitis in rats.

Factor XIII (FXIII) is the plasma-borne transglutaminase involved in fibrin clot stabilization and wound healing. FXIII levels in the plasma of patients with inflammatory bowel diseases are lower than normal and there is a significant inverse correlation of FXIII levels with clinical severity. Moreover, uncontrolled studies report beneficial effects of FXIII supplementation in patients resistant to conventional therapies. We investigated the effects of intravenous recombinant FXIII (rFXIII) treatment in experimentally induced rat colitis to verify that FXIII was the active agent in plasma FXIII concentrates and to better understand the potential therapeutic use of this protein. Colitis was induced by instillation of 12% 2.4,6-trinitrobenzenesulfonic acid (TNBS) in 50% ethanol into the colon of male Wistar rats. Rats were treated with 0.65 mg/kg rFXIII or vehicle (intravenously) daily for 10 days. Treatment was started either immediately after TNBS/EtOH instillation (to evaluate effects on developing lesions) or seven days later (to evaluate effects on established lesions). In both cases rats were killed either immediately after the end of treatment (to evaluate immediate effects) or 17 days later (to evaluate long-lasting effects). The effects of rFXIII were compared to positive (5-amino-2-hydroxybenzoic acid) control over a 35-day time course. The severity of lesions was determined by colon weight and macroscopic and histologic scores. Transglutaminase activity was measured in both colon tissue and serum. rFXIII treatment reduced lesion severity significantly not only in developing but also in established lesions. Improvements in healing persisted at least 18 days after treatment was discontinued. Serum and tissue transglutaminase levels were restored by rFXIII treatment. In conclusion, pure rFXIII is as effective as plasma FXIII concentrates in a rat model of experimental colitis. In addition, rFXIII significantly improves the healing of preexisting lesions, a characteristic useful in treatment of human inflammatory bowel diseases.

Fibrin glue for nonvariceal hemorrhage

Various endoscopic procedures are used to achieve hemostasis in peptic ulcer bleeding. All have been shown to be effective, but the rates of definitive hemostasis without rebleeding are still insufficient. We report on the efficacy and safety of fibrin glue as an injection modality for hemostasis and investigate its use for prophylactic therapy. The technique is based on separate injection of the adhesive componentss fibrinogen and thrombin that form a mechanically stable fibrin clot when they meet in the tissue. No tissue destruction or necrosis are induced. It is important to apply a sufficient number of fibrin clots near the vessel. Injection of bleeding peptic ulcer with fibrin glue has been studied as single injection and as repeated injection therapy. The demand of an effective endoscopic therapy for bleeding ulcers might be fulfilled by this method. A clinically significant benefit is shown from the use of fibrin glue as repeated application compared with the use of standard therapy with a sclerosing agent. The usefulness of control endoscopy with prophylactic retreatment of persistent bleeding stigmata is still debated. In this article, we will show how we reached the conclusion that fibrin glue injection is an effective and safe hemostatic method without tissue damage, and that repeated application probably leads to a significant improvement in rebleeding rate.

Association of the alpha-fibrinogen Thr312Ala polymorphism with poststroke mortality in subjects with atrial fibrillation.

The alpha-fibrinogen Thr312Ala polymorphism occurs in close proximity to several sites important for factor XIIIa-dependent cross-linking, which raises the possibility that it affects fibrin clot stability. We determined the association of this polymorphism with ischemic stroke, stroke subtype, and poststroke mortality. There was no significant difference in the genotype distributions of patients with acute ischemic stroke (n=519) and healthy control subjects (n=423), nor was there any association of this polymorphism with stroke subtype. In a Cox regression model, a significant interaction between Thr312Ala and atrial fibrillation was identified in relation to poststroke mortality (P=0.002). In subjects in sinus rhythm (n=418), there was no difference according to genotype in the proportion of subjects who survived (approximately 60% in each group), whereas in subjects with atrial fibrillation (n=101), there was decreased survival in those possessing the A allele (TT=42.1%, TA=18%, AA=0%). The Thr312Ala polymorphism may give rise to an increased susceptibility for embolization of intra-atrial clot, and these findings could have important implications for identifying subjects most at risk of developing thromboembolic complications.

APROTININ, FIBRIN SEALANT, AND RISK OF ANAPHYLAXIS IN CARDIAC SURGERY

Introduction. Most of anaphylactic reactions to aprotinin occur at reexposure within several months [1]. These patients generally receive intravenous high dose aprotinin, but a few react even to the low aprotinin doses contained in commercially available fibrin sealants [2]. In these sealants, aprotinin is used to extend the persistence of the fibrin clot from a few days to several weeks [3]. Our aim was to estimate the risk of anaphylaxis from aprotinin-specific antibody titers with regard to time course and application mode. Methods. Sera of 150 patients (pts.) who underwent cardiac surgery receiving first time aprotinin were sampled preoperatively, at 3.5 and 12 months postoperatively (p.o.). Aprotinin-specific IgG including all subgroups (ELISA), and aprotinin-specific IgE (fluorescence enzyme immuno assay) were analyzed. Aprotinin was given as a fibrin sealant component (n = 45; median dose: 2 ml, i.e. 3,000 KIU), systemically (n = 46; 2.0x106 KIU i.v.) and combined (n = 59; 2.0x106 KIU i.v. and 4 ml fibrin sealant, i.e. 6,000 KIU). Results. (Table 1)Table 1Total aprotinin dose was similar in antibody positive and negative patients. No aprotinin-specific antibodies were detected preoperatively. The predominant IgG-subclasses were IgG1 and IgG4. Aprotinin-specific IgE antibodies were not found postoperatively. Discussion. The presence of aprotinin-specific IgG4 indicates a risk of anaphylaxis after both application modes decreasing within one year. Fibrin sealant has the lowest potency to induce this immune response. Combined aprotinin application has the highest immunogenic potency. Due to the immunogenic potency, the benefit of aprotinin in fibrin sealants is utmostly questionable if a stable fibrin clot is necessary for a few days only. For the use of fibrin sealant as a hemostatic agent, we consider the aprotinin component dispensable.

APROTININ, FIBRIN SEALANT, AND RISK OF ANAPHYLAXIS IN CARDIAC SURGERY

Introduction. Most of anaphylactic reactions to aprotinin occur at reexposure within several months [1]. These patients generally receive intravenous high dose aprotinin, but a few react even to the low aprotinin doses contained in commercially available fibrin sealants [2]. In these sealants, aprotinin is used to extend the persistence of the fibrin clot from a few days to several weeks [3]. Our aim was to estimate the risk of anaphylaxis from aprotinin-specific antibody titers with regard to time course and application mode. Methods. Sera of 150 patients (pts.) who underwent cardiac surgery receiving first time aprotinin were sampled preoperatively, at 3.5 and 12 months postoperatively (p.o.). Aprotinin-specific IgG including all subgroups (ELISA), and aprotinin-specific IgE (fluorescence enzyme immuno assay) were analyzed. Aprotinin was given as a fibrin sealant component (n = 45; median dose: 2 ml, i.e. 3,000 KIU), systemically (n = 46; 2.0x106 KIU i.v.) and combined (n = 59; 2.0x106 KIU i.v. and 4 ml fibrin sealant, i.e. 6,000 KIU). Results. (Table 1)Table 1Total aprotinin dose was similar in antibody positive and negative patients. No aprotinin-specific antibodies were detected preoperatively. The predominant IgG-subclasses were IgG1 and IgG4. Aprotinin-specific IgE antibodies were not found postoperatively. Discussion. The presence of aprotinin-specific IgG4 indicates a risk of anaphylaxis after both application modes decreasing within one year. Fibrin sealant has the lowest potency to induce this immune response. Combined aprotinin application has the highest immunogenic potency. Due to the immunogenic potency, the benefit of aprotinin in fibrin sealants is utmostly questionable if a stable fibrin clot is necessary for a few days only. For the use of fibrin sealant as a hemostatic agent, we consider the aprotinin component dispensable.

Clinical evaluation of aprotinin

H EMOSTASIS DEPENDS on several factors: platelets, coagulation factors, blood vessel endothelium, and fibrinolysis. The coagulation cascade represents the mechanism by which a stable fibrin clot is formed (Fig 1). Most of the coagulation factors are glycoproteins synthesized in the liver. The cascade is composed of intrinsic, extrinsic, and common pathways. Factor XII, high molecular weight kininogen, prekallikrein, and factor XI form the surface activation group. Disruption in the endothelial cell layer initiates the conversion of factor XII to factor XIIa. High molecular weight kininogen together with factor XIIa, cause transformation of prekallikrein to kallikrein and activation of factor XI to factor XIa. The end product of the intrinsic pathway is factor X conversion to Xa that is catalyzed by factor IXa and VIII. Factor X, which is the junction of both pathways, starts what is called the common pathway. Activation of factor X can be achieved via the intrinsic and extrinsic pathways. The extrinsic pathways refers to the location of these factors with regard to the vasculature. The extrinsic pathway is initiated with tissue thromboplastin (factor III) that transforms factor VII to VIIa. Factor VIIa and X then act as coinitiators with the help of platelets, phospholipids, and Ca ++, to generate Factor Xa. In the common pathway, factor Xa in combination with Ca ++, phospholipids and factor Va cleaves prothrombin to produce thrombin. Fibrin (the product of thrombin cleavage of fibrinogen) is then crosslinked to form a stable clot by factor XIIIa. Platelet function is one of the most important factors in hemostasis. The platelets that are free flowing in the blood vessels initially respond to damaged or injured vessel endothelium by adhesion. On contact, platelets release several factors that propogate further platelet activation and aggregation. Once the clot is formed, some natural fibrin breakdown occurs as part of the normal healing process. Fibrinolysis is initiated by plasminogen, which is a serine protease synthesized by the liver. The degradation ofplasminogen is caused by tissue plasminogen activator (t-PA) and factor XIIa to plasmin. Plasmin splits fibrin or fibrinogen to produce fibrin or fibrinogen split products (Fig 2). There is normal local fibrinolysis wherever normal hemostasis occurs. Fibrinolysis is one of the most important causes of bleeding after cardiopulmonary bypass (CPB). A number of pharmocological strategies exist to inhibit excessive fibrinolysis. Epsilon Amino-Caproic Acid (EACA) and tranexamic acid (TA) are two synthetic antifibrinolytics that bind to plasminogen and plasmin (Fig 2) and inhibit the effect of plasmin on fibrin. Aprotinin is a serine protease inhibitor, which is isolated from bovine lung. Aprotinin was isolated and its activity then defined both as a kallikrein and a trypsin inhibitor in the 1930s. I

Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen.

Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross-linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa.

Analysis of the catalytic activity of human factor XIIIa by site-directed mutagenesis.

Factor XIIIa (FXIIIa) stabilizes fibrin clots by covalently cross-linking fibrin molecules. The purpose of this study was to determine the amino acid requirements at the active site of FXIIIa for catalysis. We selected amino acids 310-317 (Arg-Tyr-Gly-Gln-Cys-Trp-Val-Phe) in the human FXIII A-chain sequence for analysis based on the high degree of sequence homology among the different transglutaminases. We converted each amino acid in this region to Ala by site-directed mutagenesis. These recombinant FXIII A-chain mutants were expressed in Escherichia coli using the pTrc99A expression vector. FXIIIa activity was assessed by measuring the incorporation of 5-(biotinamido)pentylamine into N,N'-dimethylcasein in a solid-phase microtiter plate assay. The Cys-314-->Ala mutation yielded a recombinant protein with no FXIIIa activity. We also found that changing Gly-312 and Val-316 to Ala resulted in 22 and 65% decreases in activity, respectively. The other five mutations near the active-site Cys resulted in FXIIIa molecules in which the activity was reduced > 95%. The mechanism of SH protease catalysis is similar to transglutaminase catalysis in that both form thioester intermediates. His and Asp residues may stabilize this enzyme-substrate intermediate. Therefore, we performed site-directed mutagenesis on several His residues (His-342, His-373, and His-450) as well as Asp-396 in human FXIII. We found that changing His-342 to Ala reduced catalytic activity by 85%, while the His-373-->Ala mutant had no activity. In contrast, changing His-450 to Ala reduced FXIIIa activity by only 15%. We also examined the activity of all the mutants in a fibrin cross-linking assay. Four of the mutations (Phe-317-->Ala, Tyr-315-->Ala, Gln-313-->Ala, and Asp-396-->Ala), in which the activity toward the small primary amine was reduced by > 95%, were still capable of cross-linking the gamma-chain of fibrin. Even though these four mutants produced gamma-gamma dimers, they were not capable of forming higher molecular weight cross-linked products. Finally, we found that the binding of all the mutants to fibrin was similar to that of wild-type FXIIIa. In conclusion, we demonstrated that changing the specific amino acids Arg-310-Phe-317 to Ala substantially reduced FXIIIa activity. In addition, full catalytic activity was dependent on His-342, His-373, and Asp-396. These findings provide new insights into the catalytic mechanism of FXIIIa.

Regulation of extravascular fibrinolysis by factor XIII

The stability of fibrin clots in the extravascular compartment was examined using samples of 125I-labelled fibrin implanted subcutaneously into Balb/c mice. These samples of 125I-labelled fibrin were prepared in vitro. Crosslinking of fibrin in the presence of a high level of factor XIIIa resulted in complete conversion of γ-chain monomers to γ-γ dimers and extensive covalent polymerisation of the α-chain monomers. This fibrin, categorised as fully crosslinked (FXL), showed a 50% clot lysis time (LT-50) of 9.6±0.8 days. In contrast, fibrin, termed as partially crosslinked (PXL), prepared with a low level of Factor XIIIa, had all of its γ-chain monomers converted γ-γ dimers, but only a portion of its α-chain monomer polymerised. This PXL fibrin showed an LT-50 of 4.8±1.1 days. Non-crosslinked (NXL) fibrin displayed an LT-50 of 5.2±1.2 days. Fibrin produced by the addition of thrombin to plasma was of the PXL type and gave an LT-50 of 4.2±1.0 days. Fibrin produced by the addition of thrombin to whole blood, on the other hand, was found to be FXL and showed an LT-50 of 13.0 ± 2.0 days. Western blot analysis of fibrin samples prepared from plasma and whole blood with the use of antibody to α 2-plasmin inhibitor (α 2-PI) provided evidence that this inhibitor was covalently crosslinked to fibrin from both sources. FXL fibrin proved far more stable than either PXL fibrin or NXL fibrin to fibrinolysis by leukocyte elastase in vitro (p<0.001). Inclusion of the specific elastase inhibitor Elastatinal in the implanted NXL fibrin clot extended the LT-50 from 5 to 11.7 days (p<0.001), whereas a plasmin inhibitor, ϵ-amino caproic acid caused no change in lysis time. These results suggest that extravascular fibrinolysis is mediated chiefly by leukocyte proteases and that factor XIIIa-catalysed α-chain crosslinking is essential for protection of extravascular clots against fibrinolysis by these proteases.

Immunohistochemistry of secretory transglutaminase from rodent prostate

Transglutaminases are Ca2(+)-dependent intra- and extracellular enzymes catalyzing the cross-linking between proteins and/or polyamines, thereby eliciting divergent physiological effects such as fibrin clot stabilization or hair follicle cross-linking. A secretory transglutaminase (EC 2.3.2.13) was isolated from the coagulating gland of the rat. The protein is highly glycosylated. A fraction purified to homogeneity was used as an antigen to raise polyclonal antibodies in rabbits. These antibodies were used to identify the secretion sites of the protein within the male accessory sex glands as well as to study the immunological relationships of the respective antigen within different organs of different species. In the rat, the coagulating gland and likewise the dorsal prostate gave a positive immunoreaction. In the guinea pig, a closely related protein was detected in the anterior prostate. No cross-reactivity was found with membrane-bound transglutaminase from liver, erythrocytes or blood clotting factor XIIIa. The intraluminal secretion of the aforementioned glands was only weakly stained. No secretory granules were observed in the glandular epithelium but instead bleb-like structures reminiscent of apocrine secretion. A slight background stain of the epithelium remained even in castrated animals where secretion is largely suppressed. The background stain is attributed to a tissue-type, membrane-bound, non-secretory transglutaminase that is not androgen dependent, but instead synthesized only after androgen deprivation.

Factor XIII expression in the skin: Observations and a hypothesis

Factor XIII, a transglutaminase whose best-known function is in the coagulation cascade, is widely distributed in tissues. Three patterns of expression are found in the skin: (1) in cells in an angiocentric pattern around superficial vessels, (2) in early reparative lesions that result in fibroplasia, and (3) in certain sessile neoplasms. Around superficial vessels factor XIII probably stabilizes fibrin clots and helps to seal superficial wounds. On the basis of patterns of expression in evolving reparative lesions, factor XIII likely has an important but still undefined function in the development of fibroplasia. Factor XIII's proposed role in the promulgation of fibroplasia would be integral to the production of the stromal component of small neoplasms that is known to be associated with factor XIII expression.

Influence of factor XIII-mediated crosslinking on fibrinolysis by leukocyte elastase

Summary We have examined the effects of factor XIII-catalysed crosslinking of fibrin clots on their susceptibility to leukocyte proteinases in vitro. Fibrin clots from factor XIII-deficient plasma were lysed by leukocyte proteinase 2-fold faster than those from normal plasma. Similar differences in the rates of fibrinolysis between factor XIII-deficient and normal plasma clots were observed with purified leukocyte elastase. Non-crosslinked fibrin was lysed 2-fold faster than crosslinked fibrin. However, negligible differences in the rates of fibrinolysis by leukocyte proteinases and elastase were observed between crosslinked fibrin and fibrin with α 2 -plasmin inhibitor ( α 2 -PI) covalently attached. Analysis of the clots by PAGE in SDS during the elastase-induced lysis showed that α -monomer and α -polymers chains were fragmented first in both non-crosslinked and crosslinked fibrin. The non-crosslinked clots retained negligible quantities of degradation peptide fragments whereas the crosslinked clots contained significant amounts of larger molecular mass degradation fragments. These results suggest that the crosslinking of fibrin catalysed by factor XIIIa provides extended fibrin clot stability towards the leukocyte proteinases.

Retinoid--regulated expression of tissue transglutaminase in normal and leukemic myeloid cells.

Transglutaminases are a group of enzymes that catalyze the covalent cross-Unking of proteins by facilitating the formation of e-(γ-glutaminyl)-lysyl isopeptide bonds between protein-bound glutamine and lysine residues (1). Since the isopeptie bond is an unusually stable form of protein cross-link one may reasonably ask what is the purpose for inducing such stable bonds between polypeptide chains. In the case of the extracellular transglutaminases the answer to this question seems to be linked to the formation of mechanically stable protein clots. For instance, the seminal plasma of certain rodents contains a specific transglutaminase that cross-links seminal plasma proteins leading to the formation of a dense copulatory plug that forms in the cervical canal (2). The extensive cross-Unking of these proteins is linked to the formation of a clot that is stable for weeks. Blood plasma also contains a specific transglutaminase, plasma factor XIIIa, that is involved in the stabilization of fibrin clots. This enzyme is activated during the clotting reaction and covalently cross-links fibrin chains (3). Factor XIII also cross-links fibrin to fibronectin and possibly to collagen, a reaction that may stabilize the association of the clot with the vessel wall and platelets (4). Factor XIII also covalently incorporates A2-antiplasmin into the nascent fibrin clot (5).

Identification of normal human peripheral blood monocytes and liver as sites of synthesis of coagulation factor XIII a-chain

Factor XIII is the fibrin-stabilizing factor that covalently cross- links fibrin monomers to form a highly organized, stable fibrin clot. The plasma form of factor XIII is a heterodimer, a2b2, consisting of two a-chains and two b-chains; the intracellular form, such as in platelets and placenta, is a dimer, a2, consisting of a-chains only. The catalytic function of factor XIII, a transglutaminase, resides in the a-chain. To address questions regarding sites of synthesis of factor XIII a-chain, an EcoRI restriction fragment from the protein- coding region of the factor XIII a-chain cDNA was used as a probe for Northern blot analysis. The cDNA probe showed hybridization with a single approximately 4.0-kilobase (kb) message in poly (A)+ mRNA prepared from normal human peripheral blood monocytes and normal human liver. The results demonstrate conclusively that factor XIII a-chains are actively synthesized in circulating monocytes and in liver. To our knowledge, these data represent the first demonstration of synthesis of any blood coagulation factor in primary uncultured and unstimulated monocytes or macrophage cells.

Identification of Normal Human Peripheral Blood Monocytes and Liver as Sites of Synthesis of Coagulation Factor XIII a-Chain

Factor XIII is the fibrin-stabilizing factor that covalently cross-links fibrin monomers to form a highly organized, stable fibrin clot. The plasma form of factor XIII is a heterodimer, a2b2, consisting of two a-chains and two b-chains; the intracellular form, such as in platelets and placenta, is a dimer, a2, consisting of a-chains only. The catalytic function of factor XIII, a transglutaminase, resides in the a-chain. To address questions regarding sites of synthesis of factor XIII a-chain, an EcoRI restriction fragment from the protein-coding region of the factor XIII a-chain cDNA was used as a probe for Northern blot analysis. The cDNA probe showed hybridization with a single approximately 4.0-kilobase (kb) message in poly (A)+ mRNA prepared from normal human peripheral blood monocytes and normal human liver. The results demonstrate conclusively that factor XIII a-chains are actively synthesized in circulating monocytes and in liver. To our knowledge, these data represent the first demonstration of synthesis of any blood coagulation factor in primary uncultured and unstimulated monocytes or macrophage cells.

Platelets and Fibrinolysis

SummaryBlood platelets play a crucial role in the response of blood to vascular injury through adherence to exposed subendothelial vessel wall structures, the subsequent release of substances which trigger platelet aggregation, and release-associated changes in platelet surface properties which facilitate the interaction of coagulation factors, leading to the formation of fibrin necessary for stabilization of the initial hemostatic plug. There is substantial evidence from in vitro studies to suggest that platelets may also affect the fibrinolytic plasminogen-plasmin system thought to be one of the important hemostatic regulatory mechanisms preventing inappropriate extension of the hemostatic plug which may lead to thrombotic vessel occlusion. In this paper, the published experimental evidence concerning the effects of platelets and platelet-associated factors on the activation and function of the plasminogen-plasmin system is reviewed. The experimental findings indicate that platelets may contribute to fibrin clot stabilization through the release of substances from their storage organelles which inhibit both plasminogen activation and the action of plasmin. The physiologic and pathophysiologic significance of platelet-mediated inhibition of fibrinolysis by plasmin, as well as other potentially important plasma and cellular fibrinolytic enzymes, is still largely subject to speculation and appears to be an important area for future investigation.