scholarly journals Crotalocytin: characterization of the timber rattlesnake platelet activating protein

Blood ◽  
1980 ◽  
Vol 56 (6) ◽  
pp. 1020-1028
Author(s):  
AH Schmaier ◽  
RW Colman
Keyword(s):  
Serine Protease ◽  
Lactic Dehydrogenase ◽  
Metabolic Energy ◽  
Prostaglandin E ◽  
Amidolytic Activity ◽  
Platelet Factor ◽  
Timber Rattlesnake ◽  
Dense Granules ◽  
Rattlesnake Venom ◽  
Platelet Secretion

Crotalocytin, a platelet activating protein from timber rattlesnake venom, was studied to characterize its nature and to investigate its action on platelets. It exhibited proteolytic activity on the substrate azocoll and amidolytic activity on several peptide p-nitroanilides. The platelet activating and amidolytic activity of Crotalocytin was inhibited by diisopropylfluorophosphate. In addition, phenylmethylsulfonyl fluoride inhibited Crotalocytin's ability to stimulate platelets. Active site titration with p-nitrophenyl guanidobenzoate indicated that 52% of Crotalocytin's molecules were active and that the enzyme could also hydrolyze the titrant. These studies showed that Crotalocytin is a serine protease. Like thrombin and collagen, Crotalocytin induced simultaneous platelet aggregation and adenosine triphosphate (ATP) secretion. EDTA and prostaglandin E (PGE1) blocked Crotalocytin's ability to activate platelets; hirudin and antithrombin III did not. Crotalocytin stimulated the secretion of serotonin from dense granules and low affinity platelet factor 4 and fibrinogen from alpha-granules. Crotalocytin did not cause platelet lactic dehydrogenase loss or agglutinate formalin-fixed platelets, but it did aggregate chymotrypsin-treated platelets. Studies with antimycin A and 2 deoxy- D-glucose showed that Crotalocytin-induced platelet secretion was dependent on metabolic energy. Furthermore, Crotalocytin's induction of platelet secretion was prevented by eliminating exogenous ADP and blocking activation of the arachidonate pathway. Timber rattlesnake venom contains a serine protease that is unique, potent platelet activator.

scholarly journals Crotalocytin: characterization of the timber rattlesnake platelet activating protein

Blood ◽  
1980 ◽  
Vol 56 (6) ◽  
pp. 1020-1028 ◽  
Author(s):  
AH Schmaier ◽  
RW Colman
Keyword(s):  
Serine Protease ◽  
Lactic Dehydrogenase ◽  
Metabolic Energy ◽  
Prostaglandin E ◽  
Amidolytic Activity ◽  
Platelet Factor ◽  
Timber Rattlesnake ◽  
Dense Granules ◽  
Rattlesnake Venom ◽  
Platelet Secretion

Abstract Crotalocytin, a platelet activating protein from timber rattlesnake venom, was studied to characterize its nature and to investigate its action on platelets. It exhibited proteolytic activity on the substrate azocoll and amidolytic activity on several peptide p-nitroanilides. The platelet activating and amidolytic activity of Crotalocytin was inhibited by diisopropylfluorophosphate. In addition, phenylmethylsulfonyl fluoride inhibited Crotalocytin's ability to stimulate platelets. Active site titration with p-nitrophenyl guanidobenzoate indicated that 52% of Crotalocytin's molecules were active and that the enzyme could also hydrolyze the titrant. These studies showed that Crotalocytin is a serine protease. Like thrombin and collagen, Crotalocytin induced simultaneous platelet aggregation and adenosine triphosphate (ATP) secretion. EDTA and prostaglandin E (PGE1) blocked Crotalocytin's ability to activate platelets; hirudin and antithrombin III did not. Crotalocytin stimulated the secretion of serotonin from dense granules and low affinity platelet factor 4 and fibrinogen from alpha-granules. Crotalocytin did not cause platelet lactic dehydrogenase loss or agglutinate formalin-fixed platelets, but it did aggregate chymotrypsin-treated platelets. Studies with antimycin A and 2 deoxy- D-glucose showed that Crotalocytin-induced platelet secretion was dependent on metabolic energy. Furthermore, Crotalocytin's induction of platelet secretion was prevented by eliminating exogenous ADP and blocking activation of the arachidonate pathway. Timber rattlesnake venom contains a serine protease that is unique, potent platelet activator.

DISTINCT ROLES for Rap1b In PLATELET SECRETION and INTEGRIN aIIBb3 OUTSIDE-In SIGNALING

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2200-2200
Author(s):  
Guoying Zhang ◽  
Binggang Xiang ◽  
Ye Shaojing ◽  
Magdalena Chrzanowska-Wodnicka ◽  
Andrew J. Morris ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Critical Role ◽  
Wild Type ◽  
Clot Retraction ◽  
Platelet Factor ◽  
Dense Granules ◽  
Activation Mechanisms ◽  
Platelet Spreading ◽  
Inside Out ◽  
Platelet Secretion

Abstract Abstract 2200 Rap1b is activated by platelet agonists, and plays a critical role in integrin aIIbb3 inside-out signaling and platelet aggregation. In this study, we identify two novel functions of Rap1b in platelets. We show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that aIIbb3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Defect in secretion of Rap1b deficient platelets was not due to reduced granule contents, because the amount of serotonin (5HT) and platelet factor 4 (PF4) in Rap1b deficient platelets is similar to that in wild type platelets. Data from transmission electron microscopy indicate that under resting conditions, wild type and Rap1b deficient platelets had normal discoid shapes with similar numbers of granules. When stimulated with thrombin, wild type platelets showed an irregular appearance with protruding filopodia and lack of granules. In contrast, thrombin-stimulated Rap1b−/– platelets showed that more Rap1b−/– platelets contained visible a granules than wild type platelets. Dense granules were also more obvious in thrombin-stimulated Rap1b−/– platelets than wild type platelets. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TP deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-BAPTA. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b deficient platelets compared with wild type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b deficient platelets were not rescued by addition of MnCl2, which elicits aIIbb3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin aIIbb3 outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Vesiculation of platelets during in vitro aging

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 887-895
Author(s):  
AP Bode ◽  
SM Orton ◽  
MJ Frye ◽  
BJ Udis
Keyword(s):  
Surface Area ◽  
Platelet Activation ◽  
Lactic Dehydrogenase ◽  
Prostaglandin E ◽  
Platelet Factor ◽  
Storage Container ◽  
Fluorescent Beads ◽  
In Vitro Aging ◽  
Two Populations

Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

scholarly journals Vesiculation of platelets during in vitro aging

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 887-895 ◽  
Author(s):  
AP Bode ◽  
SM Orton ◽  
MJ Frye ◽  
BJ Udis
Keyword(s):  
Surface Area ◽  
Platelet Activation ◽  
Lactic Dehydrogenase ◽  
Prostaglandin E ◽  
Platelet Factor ◽  
Storage Container ◽  
Fluorescent Beads ◽  
In Vitro Aging ◽  
Two Populations

Abstract Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

scholarly journals Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia

2015 ◽  
Vol 396 (3) ◽  
pp. 261-275 ◽  
Author(s):  
Miroslaw Ksiazek ◽  
Abdulkarim Y. Karim ◽  
Danuta Bryzek ◽  
Jan J. Enghild ◽  
Ida B. Thøgersen ◽  
...  
Keyword(s):  
Serine Protease ◽  
Calcium Ions ◽  
Enzyme Stability ◽  
Amidolytic Activity ◽  
Tannerella Forsythia ◽  
Calcium Dependent ◽  
Genes Encoding ◽  
Mature Enzyme ◽  
Almost All ◽  
Unique Mechanism

Abstract The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37.

scholarly journals Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 869-877 ◽  
Author(s):  
Qiansheng Ren ◽  
Christian Wimmer ◽  
Michael C. Chicka ◽  
Shaojing Ye ◽  
Yi Ren ◽  
...  
Keyword(s):  
Wild Type Mouse ◽  
Limiting Factor ◽  
Attachment Protein ◽  
Altered Expression ◽  
Dense Granules ◽  
Protein Receptors ◽  
Snare Regulators ◽  
Protein Attachment ◽  
Granule Release ◽  
Platelet Secretion

Abstract Activation-dependent platelet granule release is mediated by integral membrane proteins called soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) and their regulators; however, the mechanisms for this process are ill-defined. To further characterize platelet secretion, we analyzed the function of platelets from Unc13dJinx mice. Platelets from these animals lack the putative vesicle priming factor, Munc13-4, and have a severe secretion defect. Release from dense granules was completely ablated and that from α-granules and lysosomes was severely compromised. Unc13dJinx platelets showed attenuated aggregation and, consequently, Unc13dJinx mice had prolonged tail-bleeding times. The secretion defect was not due to altered expression of SNAREs or SNARE regulators, defective granule biogenesis, or faulty platelet activation. The defective release could be rescued by adding recombinant Munc13-4 to permeabilized Unc13dJinx platelets. In wild-type mouse platelets, Munc13-4 levels were lower than those of SNAREs suggesting that Munc13-4 could be a limiting component of the platelets' secretory machinery. Consistently, Munc13-4 levels directly correlated with the extent of granule release from permeabilized platelets and from intact, heterozygous Unc13dJinx platelets. These data highlight the importance of Munc13-4 in platelets and indicate that it is a limiting factor required for platelet secretion and hemostasis.

Effects Of Short Time High Shear Exposure Upon Platelet Function And The Coagulation System Under Heparin Anticoagulation

1981 ◽  
Author(s):  
L J Wurzinger ◽  
R Opitz ◽  
P Blasberg ◽  
K Bialonski ◽  
H Schmid-Schönbein
Keyword(s):  
Platelet Function ◽  
Lactic Dehydrogenase ◽  
Arterial Stenosis ◽  
Coagulation System ◽  
Shear Stresses ◽  
High Shear ◽  
Platelet Factor ◽  
Shear Rates ◽  
Heparin Anticoagulation ◽  
Short Time

The fact that high shear activates and damages platelets has been suspected to be a major cause of thromboembolism in artificial internal organs (AIO) or in arterial stenosis. In AIO wall shear stresses well above 50 Nm-2 have been computed to which blood cells are exposed for times in the order of milliseconds (ms). Unfortunately, the studies on this subject employing defined flow conditions operate with exposure times higher than 10 seconds. The pupose of the present study was to elucidate the effects of high shear exposure for ms upon platelet function (ADP induced platelet aggregation (PA), platelet procoagulant activity {PF- 3)) under heparin anticoagulation, which is also used in AIO.To apply shear rates ranging from 50 - 220 Nm-2 to heparinized PRP for defined exposure times between 7 - 700 ms a flow through Couette-viscometer was employed. Platelet factor 3 (PF-3) availability was estimated by using a modified Stypven time technique. Lactic dehydrogenase (LDH) liberation was taken as a measure for platelet destruction. All steps of the experimental procedure were carried out at 37°C.From our data we conclude that, in the presence of physiological calcium levels (heparin anticoagulation) shear stresses and exposure times that certainly occur in AIO are able to activate platelets and procoagulant potential of blood.

scholarly journals Inherited abnormalities in platelet organelles and platelet formation and associated altered expression of low molecular weight guanosine triphosphate-binding proteins in the mouse pigment mutant gunmetal

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2626-2635 ◽  
Author(s):  
RT Swank ◽  
SY Jiang ◽  
M Reddington ◽  
J Conway ◽  
D Stephenson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Binding Proteins ◽  
Low Molecular Weight ◽  
Guanosine Triphosphate ◽  
Platelet Factor ◽  
Altered Expression ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding ◽  
Platelet Formation

Abstract Gunmetal (gm/gm) is a recessively inherited mouse pigment dilution mutant that has high mortality and poor reproductive rates. In these studies, several hematologic defects were found associated with the mutation, including prolonged bleeding times, together with thrombocytopenia and increased platelet size. A unique feature is the presence of simultaneous abnormalities in two platelet organelles, dense granules and alpha-granules. The dense granule component serotonin is present at about half the normal concentration, as are visible dense granules. Three alpha-granule components (fibrinogen, platelet factor 4, and von Willebrand factor) are also significantly reduced. Thus, in several respects the gunmetal mutant resembles the human gray platelet syndrome. A novel abnormality in expression of low molecular weight guanosine triphosphate (GTP)-binding proteins occurs in platelets of gunmetal. In Western blot assays, two additional GTP- binding proteins of 28.5 and 25 Kd were detected. The abnormal expression of GTP-binding proteins is, like the hematologic defects, genetically recessive and is tissue specific. Liver, kidney, brain, spleen, macrophages, and neutrophils have normal GTP-binding protein expression. The additional GTP-binding proteins are soluble. The data indicate that platelet formation and platelet organelle biogenesis are under common genetic control and that abnormal regulation of GTP- binding proteins may affect one or both processes.

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