Role of Sialic Acid for Platelet Lifespan and Function

Abstract Although sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet half-life is not fully clarified. We and others have previously provided evidence that thrombocytopenia in mice deficient in the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice) is caused by rapid clearance of the platelets due to recognition of surface galactose by asialoglycoprotein receptor-expressing scavenger cells. Here we report new insight into clearance mechanisms, activation and production of sialic acid deficient platelets. Immunofluorescence staining of organ specimens harvested shortly after platelet transfusion demonstrated the predominant clearance of ST3Gal-IV−/− platelets by liver macrophages and, as previously reported, hepatocytes. The differential cellular clearance pathways were further explored following macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that restored ST3Gal-IV−/− platelet circulation to approximately 40% of normal values, confirming that macrophages play a major role in the clearance of sialic acid deficient platelets and that other clearance pathways are equally important. Ingestion of ST3Gal-IV−/− platelets by hepatocytes was confirmed by in vitro HepG2 phagocytosis assays. We next investigated the function of desialylated platelets. Platelet binding of von Willebrand factor (vWf) upon botrocetin stimulation was 3 fold higher for ST3Gal-IV−/− platelet rich plasma compared to ST3Gal- IV+/+ platelet rich plasma. The circulation time of wild-type platelets transfused into ST3Gal-IV−/− mice was 20% reduced compared to control platelets, indicating that platelet removal could be accelerated due to binding of desialylated vWf to platelets. Loss of sialic acid did not affect platelet production in vitro and in vivo as cultivated megakaryocytes was found to produce proplatelets normally and measurements of reticulated platelets by flow cytometry showed a 3-fold increased thrombocytopoietic activity in the ST3Gal- IV−/− mice compared to wild-type mice. Interestingly, thiazole orange staining revealed a significant negative correlation between platelet size and platelet age for both genotypes, In conclusion, depletion of the ST3Gal-IV gene promotes binding of vWf to platelets, exposes platelet surface galactose residues to the lectin domain of asialoglycoprotein receptors on both hepatocytes and liver macrophages, resulting in rapid platelet clearance from the circulation and supporting previous findings that platelets decrease in size while aging in circulation.

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Platelets Lacking Sialic Acid Clear Rapidly from the Circulation Due to Ingestion by Asialoglycoprotein Receptor-Expressing Liver Macrophages and Hepatocytes.

Blood ◽

10.1182/blood.v108.11.1521.1521 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1521-1521

Author(s):

Anne Louise Soerensen ◽

Hans H. Wandall ◽

Sunita Patel ◽

Jennifer Richardson ◽

Joseph Italiano ◽

...

Keyword(s):

Sialic Acid ◽

Kupffer Cells ◽

Asialoglycoprotein Receptor ◽

Receptor Expression ◽

Wild Type ◽

Platelet Surface ◽

Platelet Recovery ◽

Lectin Domain ◽

Surface Receptor

Abstract Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet survival is unclear. We investigated the importance of sialic acid for platelet clearance using mice deficient in the ST3GalIV sialyltransferase gene (ST3GalIV−/− mice) and identified a novel clearance mechanism previously unrecognized for platelets. ST3GalIV catalyzes the addition of sialic acid onto exposed galactose residues of cell surface glycoproteins. ST3GalIV−/− mice have increased platelet surface galactose exposure, a 70% reduction in platelet count, and prolonged bleeding times. We report that ST3GalIV−/− platelets transfused into wild-type C57BL/6J mice exhibit markedly reduced recoveries and shortened survivals respectively compared to littermate wild-type platelets. Infusion of asialofetuin, an antagonist of the asialoglycoprotein-receptor (ASGPR) restored platelet recovery time and initial circulation of ST3GalIV−/− platelets to normal values. Immunohistochemical studies of organ specimens harvested shortly after transfusion of biotin-labelled platelets demonstrated the predominant clearance of ST3GalIV−/− platelets by the liver Kupffer cells and, unexpectedly, hepatocytes. Megakaryocytes cultured from ST3GalIV−/− mice produced proplatelets normally compared to megakaryocytes generated from wild-type littermates, indicating that the thrombocytopenia in the ST3GalIV−/− mice is not due to reduced platelet production. Comparison of ST3GalIV+/+ and ST3GalIV−/− platelet surface receptor expression as evidenced by flow cytometry and preliminary in vitro activation studies did not reveal any significant differences in the two genotypes. We conclude that the absence of terminal sialic acid residues on platelet surfaces exposes galactose residues to the lectin domain of ASGPR on both hepatocytes and liver Kupffer cells, resulting in platelet clearance from the circulation.

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PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets

Thrombosis and Haemostasis ◽

10.1160/th07-03-0207 ◽

2007 ◽

Vol 98 (10) ◽

pp. 806-812 ◽

Cited By ~ 46

Author(s):

Vandana Dole ◽

Wolfgang Bergmeier ◽

Ian Patten ◽

Junichi Hirahashi ◽

Tanya Mayadas ◽

...

Keyword(s):

Endothelial Activation ◽

Leukocyte Rolling ◽

Wild Type ◽

Platelet Surface ◽

Activated Platelets ◽

And Function ◽

Body Secretion

SummaryWe have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-1 and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1-/- compared to wild-type mice. The leukocyte integrin αMβ2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte- neutrophil elastase,known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium andWeibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet.

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Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽

10.1182/blood.v114.22.2531.2531 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2531-2531

Author(s):

Pani A. Apostolidis ◽

Stephan Lindsey ◽

William M. Miller ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Platelet Surface ◽

Platelet Counts ◽

Reticulated Platelets ◽

High Ploidy ◽

And Control ◽

Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

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TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

Blood ◽

10.1182/blood-2021-152975 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1050-1050

Author(s):

Angela Doerr ◽

Denise Pedrosa ◽

Maria Schander ◽

Yotis A. Senis ◽

Alexandra Mazharian ◽

...

Keyword(s):

Platelet Activation ◽

Type I Collagen ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Type I ◽

Wild Type ◽

Platelet Surface ◽

Knock Out ◽

Integrin Αiibβ3

Abstract Background Thrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors. Aim We aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation. Methods Recombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma. Results VWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1. Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice. Conclusions This study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. Disclosures Ruf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

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Detection of Activated Feline Platelets in Platelet-rich Plasma by Use of Fluorescein-labeled Antibodies and Flow Cytometry

Veterinary Pathology ◽

10.1177/030098589403100507 ◽

1994 ◽

Vol 31 (5) ◽

pp. 553-560 ◽

Cited By ~ 14

Author(s):

E. G. Welles ◽

C. Bourne ◽

J. W. Tyler ◽

M. K. Boudreaux

Keyword(s):

Platelet Rich Plasma ◽

Flow Cytometric Analysis ◽

Trisodium Citrate ◽

Serotonin Release ◽

Prostaglandin E ◽

Human Fibrinogen ◽

Platelet Surface ◽

Flow Cytometric

Platelets contribute to prethrombotic or thrombotic states; however, accepted evaluation methods (i.e., in vitro testing by use of an aggregometer) of platelet function in cats can be difficult because of the large volume of blood required from which platelets are isolated and the potential for platelet activation due to difficult venipunctures in sometimes uncooperative or excited animals. The activation problem also contributes to errors in platelet counts. Platelets from four domestic short haired cats (two males, two females, 2–3 years old) minimally restrained without sedation or anesthesia were evaluated. Blood (5 ml) was collected by jugular venipuncture directly into syringes containing 3.8% trisodium citrate (nine parts blood to one part anticoagulant) plus prostaglandin E1 (3 μM; 0.25, 0.5, 1, or 2 μl/500 μl citrate) or 3.8% trisodium citrate alone. Prostaglandin E1, which is a stable metabolite of arachidonic acid with platelet inhibitory properties similar to those of prostaglandin I2 was added to the anticoagulant to prevent activation of platelets during the collection process. Feline platelets exposed to prostaglandin E1 became immediately and persistently nonreactive to agonists, which negated their use in functional studies (aggregation, 14C-serotonin release, binding of fluorescein-conjugated antifibrinogen) but improved platelet counting accuracy. Detection of in vivo activation of platelets in prethrombotic and thrombotic states in humans has been done by identification of activation-dependent molecules on platelet surfaces by use of specific antibody recognition and detection by flow cytometric analysis. Many activation-dependent platelet surface receptor changes are species specific; however, fibrinogen appears to be conserved across species. Results of flow cytometric analysis by forward and side angle light scatter easily identified feline platelets in 10 μl of platelet-rich plasma but were less definitive in identification of platelets in whole blood. Feline platelets in platelet-rich plasma activated by ADP (5 or 50 μM), which allowed binding of fibrinogen to exposed fibrinogen receptors, were labeled with fluorescein-conjugated anti-human fibrinogen. Labeled platelets were identified as a separate population by flow cytometric staining pattern and fluorescence intensity from nonlabeled platelets or nonspecifically-labeled resting platelets. This technique should allow detection of activated feline platelets from extremely small quantities of blood, which could facilitate repetitive and serial platelet testing in diseases thought to be accompanied, caused, or augmented by thrombotic conditions.

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Surface Sialic Acid Prevents Loss of GPIbα during Platelet Storage and Rescues In Vivo Survival of Murine Platelets.

Blood ◽

10.1182/blood.v110.11.138.138 ◽

2007 ◽

Vol 110 (11) ◽

pp. 138-138 ◽

Cited By ~ 3

Author(s):

Gerard Jansen ◽

Emma C. Josefsson ◽

John H. Hartwig ◽

Karin M. Hoffmeister

Keyword(s):

Flow Cytometry ◽

Sialic Acid ◽

Shape Change ◽

Platelet Surface ◽

Integrin Αiibβ3 ◽

Surface Receptors ◽

Platelet Survival ◽

Platelet Receptor

Abstract Platelet processing and storage are associated with platelet lesion, e.g. shape change, activation, release reaction and apoptosis, which is partially due to loss of surface receptors. Surface sialic acid is considered to be a key determinant for the survival of circulating blood cells and glycoproteins. However, its role in platelet receptor loss and platelet survival is unclear. In this study, the relationship between surface sialic acid and platelet receptor loss was investigated in vitro and in vivo. Murine platelets stored at room temperature for 6 hours lost surface sialic acid, as evidenced by flow cytometry using FITC conjugated RCA I lectin, which recognizes exposed galactose residues. This loss correlated with a 30–60% loss of surface receptors GPIbα and GPV, but not GPIX and integrin αIIbβ3, as measured by flow cytometry. Treatment of murine platelets with the neuraminidase (NA) substrate fetuin partially decreases the loss of GPIbα and GPV to 10–20%. In vitro, sialic acid was cleaved from the platelet surface by adding NA (α2-3,6,8-NA (V. cholerae) or α2-3,6,-NA (C. perfringens)) to murine platelets. Removal of sialic acid correlated with the removal of 50–60% of surface GPIbα and GPV, but not GPIX and integrin αIIbβ3. Addition of fetuin, or the more specific NA inhibitor 2,3-dehydro-2-deoxy-, sodium salt (DANA), completely prevented this loss, as determined by both flow cytometry and Western blot analysis. Murine platelets treated with α2-3,6,8-NA (V. cholerae) ± the addition of DANA were labeled with the green dye CMFDA and transfused into age-, strain- and sex-matched C57BL/6 mice to measure platelet survival. NA-treated platelets were cleared within minutes after transfusion, whereas the addition of DANA rescued platelet survival to control-count increments. Our study shows that inhibiting the loss of surface sialic acid prevents platelet surface GPIbα and GPV loss during storage in vitro and rescues platelet survival in vivo.

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Loss Of Platelet Membrane Glycoproteins Or Terminal Sialic Acid Detected By Fitc-Lectins

10.1055/s-0038-1653289 ◽

1981 ◽

Author(s):

L McGregor ◽

J L McGregor ◽

K J Clemetson ◽

M Dechavanne ◽

E F Lüscher

Keyword(s):

Sialic Acid ◽

Ricinus Communis ◽

Lectin Binding ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Binding Studies ◽

Platelet Surface ◽

Terminal Sialic Acid

Pre-thrombic conditions in certain individuals resulting from enhanced platelet-vessel wall or platelet-platelet interactions are perhaps characterized by a reduction in certain membrane glycoproteins or loss of terminal sialic acid. In order to investigate if such changes are detectable, the binding of FITC-lectins to human platelets treated under in vitro conditions with certain proteases to mimic possible in vivo changes occuring on the platelet surface, has been examined. Human platelets were isolated, washed and either treated with neuraminidase (10 U) or plasmin (1 CU) before fixing with formaldehyde. Binding studies were performed by the method of Monsigny et al. using FITC labelled wheat germ agglutinin (WGA), Lens culinaris lectin (LCL), Ricinus communis agglutinin (RCA) and concanavalin A (ConA). The number of lectin-binding sites (n) and the dissociation constant (Kd) were obtained by Steck and Wallach reciprocal plots. After neuraminidase or plasmin treatment n was reduced but Kd remained approximately the same with WGA. FITC-RCA-60 gave a slight fluorescence with untreated and very strong fluorescence with neuraminidase treated platelets. Platelet glycoproteins separated by 2-dimensional gel electrophoresis were identified by binding of fluorescent lectins. Plasmin decreased the intensity of GP Ib and IIb and removed Ia completely. Neuraminidase decreased the labelling of Ib by WGA. These techniques show promise as methods of detecting pre-thrombotic conditions.

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Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647788 ◽

1973 ◽

Vol 29 (02) ◽

pp. 490-498 ◽

Cited By ~ 6

Author(s):

Hiroh Yamazaki ◽

Itsuro Kobayashi ◽

Tadahiro Sano ◽

Takio Shimamoto

Keyword(s):

Platelet Count ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

The Other ◽

Endothelial Surface ◽

Important Interaction ◽

Blood Coagulability ◽

The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

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Storage of Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647709 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 405-416 ◽

Cited By ~ 3

Author(s):

M. R Hardeman ◽

Carina J L. Heynens

Keyword(s):

Storage Temperature ◽

Platelet Rich Plasma ◽

Blood Platelets ◽

Storage Period ◽

Serotonin Uptake ◽

Hypotonic Shock ◽

Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

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A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650089 ◽

1980 ◽

Vol 44 (02) ◽

pp. 081-086 ◽

Cited By ~ 8

Author(s):

C V Prowse ◽

A E Williams

Keyword(s):

Platelet Rich Plasma ◽

Thrombus Formation ◽

Factor Ix ◽

Coagulation System ◽

In Vitro Tests ◽

Clinical Use ◽

Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

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