[20] Quantifying thrombin-catalyzed release of fibrinopeptides from fibrinogen using high-performance liquid chromatography

Ng, Assunta S.; Lewis, Sidney D.; Shafer, Jules A.

doi:10.1016/0076-6879(93)22023-9

Cited by 40 publications

(45 citation statements)

References 46 publications

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“…34,35 Briefly, fibrinogen (0.1 mg/mL) in HBS, with or without 1 mM CaCl 2 , was incubated with human ␣-thrombin (0.005 U/mL) at ambient temperature and the reactions stopped by incubation at 100°C. Fibrinopeptides were separated by HPLC and their concentrations determined from the peak areas measured with the ISCO Chemresearch 2.4 software.…”

Section: Thrombin-catalyzed Fibrinopeptide Releasementioning

confidence: 99%

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Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Okumura

Gorkun²,

Terasawa³

et al. 2004

Blood

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Crystallographic structures indicate that ␥-chain residue Asn308 participates in D:D interactions and indeed substitutions of ␥Asn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: ␥Asn308 changed to lysine (␥N308K), isoleucine (␥N308I), and alanine (␥N308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa-catalyzed crosslinking by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of ␥N308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with ␥N308K. Factor XIIIa-catalyzed ␥-␥ dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) ␥-␥ dimer formation of only ␥N308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both. IntroductionFibrinogen is a 340-kDa glycoprotein that circulates in the blood at 5.88 to 11.76 M (2-4 g/L). It is composed of a pair of 3 polypeptide chains, A␣, B␤, and ␥, connected by 29 intrachain and interchain disulfide bonds. 1 The 6 chains are arranged into 3 globular nodules. The central E nodule contains the N-termini of all chains and the distal D nodules contain the C-termini of the ␤, ␥, and a short segment of the ␣ chains. Coiled coils composed of all 3 chains link the E and D nodules. 2 The C-terminal residues of A␣ chains form globular nodules that are associated with one another, as well as with the E nodule. 3 Recent crystallographic studies have provided high-resolution structures of the C-terminal region of the ␥ chain, which contains functional sites for fibrin polymerization, calcium binding, platelet aggregation, and factor XIII (FXIII) cross-linking. [4][5][6] During coagulation, thrombin cleaves fibrinogen, releasing fibrinopeptides A (FpA) and B (FpB) from the N-termini of the A␣ and B␤ chains, respectively, and converting fibrinogen to fibrin monomers (for reviews, see Blomback 1 and Doolittle 2 ). Fibrin monomers polymerize spontaneously through a 2-step process, with a second step being calcium-dependent. 7,8 The first step begins with the release of FpA. This exposes the "A" site, which interacts with the "a" site in the D nodule of another molecule, leading to the formation of half-staggered, double-stranded protofibrils. 9,10 Within protofibrils, individual fibrin mol...

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Section: Thrombin-catalyzed Fibrinopeptide Releasementioning

confidence: 99%

“…Specificity constants, k cat /K m , were determined as described. 34,35 Reactions were performed in triplicate. …”

mentioning

confidence: 99%

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Okumura

Gorkun²,

Terasawa³

et al. 2004

Blood

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“…on May 12, 2018. by guest www.bloodjournal.org From release, as is standard for fibrinogen reactions and has been described previously. 16 However, we were unable to accurately fit the FpB release data to the conventional equation describing FpB release; thus, the FpB data in Figure 6 are not fit to any equation. It should be noted that the calculated rate constants (k2) from these curves showed that the rate of FpB release from STAT5-deficient mice (1.04 Ϯ 0.08 minutes Ϫ1 ) was 2.3-fold faster than control mice (0.45 Ϯ 0.05 minutes Ϫ1 ).…”

Section: Increased Thrombosis In Stat5-deficient Mice 5729mentioning

confidence: 95%

“…Normalized data were fit with a simple first-order reaction to determine rate constants for release of FpA (k1), as described. 16 Each FpB dataset was normalized to its calculated FpA max. Normalized data were plotted together, and while a fit was attempted, no acceptable fit was made.…”

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confidence: 99%

Increased thrombosis susceptibility and altered fibrin formation in STAT5-deficient mice

Nordstrom

Holliday

Sos

et al. 2010

Blood

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To explore the effect(s) of growth hormone signaling on thrombosis, we studied signal transduction and transcription factor 5 (STAT5)-deficient mice and found markedly reduced survival in an in vivo thrombosis model. These findings were not explained by a compensatory increase in growth hormone secretion. There was a modest increase in the activity of several procoagulant factors, but there was no difference in the rate or magnitude of thrombin generation in STAT5-deficient mice relative to control.However, thrombin-triggered clot times were markedly shorter, and fibrin polymerization occurred more rapidly in plasma from STAT5-deficient mice. Fibrinogen depletion and mixing studies indicated that the effect on fibrin polymerization was not due to intrinsic changes in fibrinogen, but resulted from changes in the concentration of a circulating plasma inhibitor. While thrombin-triggered clot times were significantly shorter in STAT5-deficient animals, reptilase-triggered clot times were unchanged. Accordingly, while the rate of thrombin-catalyzed release of fibrinopeptide A was similar, the release of fibrinopeptide B was accelerated in STAT5-deficient plasma versus control. Taken IntroductionThrombosis-related diseases such as myocardial infarction, stroke, and venous thromboembolism account for substantial morbidity and mortality worldwide. We recently demonstrated that thrombosis susceptibility is mediated, in part, by sex-specific patterns of growth hormone (GH) secretion and subsequent effects on expression of coagulation-related genes in the liver. 1 GH is a pleiotropic hormone that is synthesized and secreted by the anterior pituitary gland and has diverse effects on its target tissues. 2,3 Hepatic GH signaling occurs via the type I cytokine receptor, growth hormone receptor, and activation of its primary downstream effectors, the Janus kinase 2 (JAK2) and signal transduction and transcription factor 5 (STAT5). Mice deficient in both STAT5A and STAT5B are largely insensitive to GH and consequently, have an impaired insulin-like growth factor 1-mediated feedback inhibition and dysregulated GH secretion. [4][5][6][7][8] STAT5 is also an integral component of interleukin, erythropoietin, and prolactin signaling pathways. Furthermore, STAT5 can be activated independently of JAK2 by receptor tyrosine kinases and other mechanisms. 9,10 STAT5 has thus been shown to be a central component of GH signaling, as well as other important cytokine signaling pathways.Previously, we demonstrated that GH-deficient little mice are protected from thrombosis in vivo. 1 Considering the important role of STAT5 in GH signaling, we aimed to study the role of STAT5 in thrombosis. In this study, we characterized 2 mouse models of STAT5 deficiency. We predicted that mice deficient in STAT5 would have a decrease in thrombosis susceptibility, as seen in GH-deficient little mice. Surprisingly, we found that the genetic loss of STAT5 resulted in increased susceptibility to thrombosis, in vivo, and shortened clotting times, in vitro. These...

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“…The fibrinopeptide release curves were plotted using Kinetic Equations 1 and 2, described by Ng et al (Ng et al, 1993). In both equations, e represented thrombin concentration and t the reaction time.…”

Section: High Performance Liquid Chromatography (Hplc)mentioning

confidence: 99%

Manipulating fibrin structure of hematomas enhances large bone defects healing

Wang¹

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[20] Quantifying thrombin-catalyzed release of fibrinopeptides from fibrinogen using high-performance liquid chromatography

Cited by 40 publications

References 46 publications

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Increased thrombosis susceptibility and altered fibrin formation in STAT5-deficient mice

Manipulating fibrin structure of hematomas enhances large bone defects healing

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