scholarly journals Microfluidic Assay of Platelet Deposition on Collagen by Perfusion of Whole Blood from Healthy Individuals Taking Aspirin

2013 ◽  
Vol 59 (8) ◽  
pp. 1195-1204 ◽  
Author(s):  
Ruizhi Li ◽  
Susanne Fries ◽  
Xuanwen Li ◽  
Tilo Grosser ◽  
Scott L Diamond
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Microfluidic Devices ◽  
Fibrillar Collagen ◽  
Healthy Individuals ◽  
Shear Rates ◽  
Platelet Deposition ◽  
Wall Shear ◽  
Aspirin Ingestion ◽  
Venous Wall

BACKGROUND Microfluidic devices can create hemodynamic conditions for platelet assays. We validated an 8-channel device in a study of interdonor response to acetylsalicylic acid (ASA, aspirin) with whole blood from 28 healthy individuals. METHODS Platelet deposition was assessed before treatment or 24 h after ingestion of 325 mg ASA. Whole blood (plus 100 μmol/L H-d-Phe-Pro-Arg-chloromethylketone to inhibit thrombin) was further treated ex vivo with ASA (0–500 μmol/L) and perfused over fibrillar collagen for 300 s at a venous wall shear rate (200 s−1). RESULTS Ex vivo ASA addition to blood drawn before aspirin ingestion caused a reduction in platelet deposition [half-maximal inhibitory concentration (IC50) approximately 10–20 μmol/L], especially between 150 and 300 s of perfusion, when secondary aggregation mediated by thromboxane was expected. Twenty-seven of 28 individuals displayed smaller deposits (45% mean reduction; range 10%–90%; P < 0.001) from blood obtained 24 h after ASA ingestion (no ASA added ex vivo). In replicate tests, an R value to score secondary aggregation [deposition rate from 150 to 300 s normalized by rate from 60 to 150 s] showed R < 1 in only 2 of 28 individuals without ASA ingestion, with R > 1 in only 3 of 28 individuals after 500 μmol/L ASA addition ex vivo. At 24 h after ASA ingestion, 21 of 28 individuals displayed poor secondary aggregation (R < 1) without ex vivo ASA addition, whereas the 7 individuals with residual secondary aggregation (R > 1) displayed insensitivity to ex vivo ASA addition. Platelet deposition was not correlated with platelet count. Ex vivo ASA addition caused similar inhibition at venous and arterial wall shear rates. CONCLUSIONS Microfluidic devices quantified platelet deposition after ingestion or ex vivo addition of aspirin.

scholarly journals Evidence that fibrin alpha-chain RGDX sequences are not required for platelet adhesion in flowing whole blood

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1001-1009 ◽  
Author(s):  
RR Hantgan ◽  
SC Endenburg ◽  
JJ Sixma ◽  
PG de Groot
Keyword(s):  
Whole Blood ◽  
Platelet Adhesion ◽  
Tissue Type ◽  
Shear Rates ◽  
Putative Receptor ◽  
Type Plasminogen Activator ◽  
Platelet Deposition ◽  
Wall Shear ◽  
Adhesive Interactions

Abstract The role of the RGDX putative receptor-recognition sites, which are present on the alpha chains of fibrin, in promoting platelet adhesion has been examined in flowing whole blood using the rectangular perfusion chamber at wall shear rates of 340 and 1,600/s. Platelets adhered to a comparable extent to surfaces coated with native fibrin and surfaces coated with fragment X-fibrin, a product of limited fibrinolysis that lacks the RGDS sites normally present at positions 572 to 575 of the alpha chains. The strengths of these adhesive interactions were comparable based on the concentrations of the antiadhesive peptide D-RGDW required to block platelet deposition to native and fragment X-fibrin at both low and high wall shear rate. Blocking either or both RGDX sequences with peptide-specific monoclonal antibodies did not inhibit platelet deposition in perfusion experiments performed with normal blood at 340/s, indicating that neither RGD motif is required for adhesion. However, adhesion was partly inhibited by anti-RGDX antibodies when perfusions were performed with blood from an afibrinogenemic patient, suggesting the RGDX sequences may play a limited role in platelet deposition. Exposure of fibrin surfaces to plasminogen/tissue-type plasminogen activator did cause a time- dependent loss of adhesiveness, but this effect was only weakly correlated with proteolysis of the fibrin alpha chains. These observations provide evidence that neither RGDX sequence is required for platelets to adhere avidly to fibrin in flowing blood. These results further suggest that incomplete fibrinolysis yields a highly thrombogenic surface.

scholarly journals Evidence that fibrin alpha-chain RGDX sequences are not required for platelet adhesion in flowing whole blood

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1001-1009
Author(s):  
RR Hantgan ◽  
SC Endenburg ◽  
JJ Sixma ◽  
PG de Groot
Keyword(s):  
Whole Blood ◽  
Platelet Adhesion ◽  
Tissue Type ◽  
Shear Rates ◽  
Putative Receptor ◽  
Type Plasminogen Activator ◽  
Platelet Deposition ◽  
Wall Shear ◽  
Adhesive Interactions

The role of the RGDX putative receptor-recognition sites, which are present on the alpha chains of fibrin, in promoting platelet adhesion has been examined in flowing whole blood using the rectangular perfusion chamber at wall shear rates of 340 and 1,600/s. Platelets adhered to a comparable extent to surfaces coated with native fibrin and surfaces coated with fragment X-fibrin, a product of limited fibrinolysis that lacks the RGDS sites normally present at positions 572 to 575 of the alpha chains. The strengths of these adhesive interactions were comparable based on the concentrations of the antiadhesive peptide D-RGDW required to block platelet deposition to native and fragment X-fibrin at both low and high wall shear rate. Blocking either or both RGDX sequences with peptide-specific monoclonal antibodies did not inhibit platelet deposition in perfusion experiments performed with normal blood at 340/s, indicating that neither RGD motif is required for adhesion. However, adhesion was partly inhibited by anti-RGDX antibodies when perfusions were performed with blood from an afibrinogenemic patient, suggesting the RGDX sequences may play a limited role in platelet deposition. Exposure of fibrin surfaces to plasminogen/tissue-type plasminogen activator did cause a time- dependent loss of adhesiveness, but this effect was only weakly correlated with proteolysis of the fibrin alpha chains. These observations provide evidence that neither RGDX sequence is required for platelets to adhere avidly to fibrin in flowing blood. These results further suggest that incomplete fibrinolysis yields a highly thrombogenic surface.

Measurement of Blood-Borne Tissue Factor Procoagulant Activity in Healthy Individuals and Cancer Patients Using a Novel Assay.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1938-1938
Author(s):  
Rachel E. Tilley ◽  
Todd Holscher ◽  
Rajesh Belani ◽  
Jorge Nieva ◽  
Nigel Mackman
Keyword(s):  
Cancer Patients ◽  
Tissue Factor ◽  
Whole Blood ◽  
Time Course ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Procoagulant Activity ◽  
Functional Assay ◽  
Healthy Individuals ◽  
Stimulation Of

Abstract Tissue factor (TF) is the cellular initiator of the extrinsic pathway of coagulation. Recent studies have reported circulating blood-borne TF (BBTF) antigen by methods including ELISA and flow cytometry. Whether this BBTF is active has been rarely addressed due to the lack of a simple functional assay. We have developed a new assay that measures BBTF procoagulant activity. Whole blood was collected into tubes containing citrate and corn trypsin inhibitor (FXIIa inhibitor). First, we compared the procoagulant activity obtained in detergent lyzed platelets and microparticles with peripheral blood mononuclear cells (PBMCs) following a 5 hour ex vivo stimulation of whole blood with LPS. Platelets and microparticles had 3% (23 ± 10 mU/mL) and 0.1% (1.0 ± 0.4 mU/mL), respectively, of the procoagulant activity of PBMCs (1048 ± 200 mU/mL). The procoagulant activity of PBMCs and platelets was inhibited by >90% in the presence of an anti-TF polyclonal antibody. Next, we determined the time course of TF-dependent procoagulant activity of microparticles and platelets after ex vivo LPS stimulation of whole blood. In both cases, a dramatic increase in microparticle and platelet procoagulant activity was observed after 12 hours of LPS stimulation, with a maximum activity observed at 48 hours (48 mU/mL for microparticles and 1028 mU/mL for platelets at 48 hours). The majority (>90%) of this procoagulant activity was TF-dependent. Therefore, we subsequently analyzed combined platelet and microparticle (P+MP) fractions for BBTF activity. We determined functional BBTF activity in P+MP fractions from the blood of healthy individuals with and without LPS ex vivo stimulation. In healthy individuals, very low levels of BBTF procoagulant activity was detected in the absence of LPS stimulation (0.22 ± 0.09 mU/mL) and this activity was decreased on average by 28% with an anti-TF antibody (0.16 ± 0.1 mU/mL; p = 0.01). In contrast, P+MP from LPS stimulated blood demonstrated on average 30 fold higher procoagulant activity, of which >90% was TF-dependent. Cancer is associated with an increased susceptibility to develop pathological thrombosis. Using our new assay, we demonstrate a significant elevation of the procoagulant activity of P+MP fractions from advanced (stage IV) solid tumor patients of varying histology’s (n=14; 0.88 ± 0.55 mU/mL) compared with normal subjects (p <0.001). Furthermore, we showed that on average 50% of the procoagulant activity was TF-dependent (procoagulant activity was reduced to 0.57 ± 0.35 mU/mL in the presence of an anti-TF antibody; p < 0.01), suggesting that circulating BBTF in cancer patients has the potential to contribute to thrombosis in vivo. In summary, we have developed a novel assay that measures BBTF activity. This assay may be useful in the detection of a pre-thrombotic state in cancer patients.

scholarly journals Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 345-353 ◽  
Author(s):  
RR Hantgan ◽  
G Hindriks ◽  
RG Taylor ◽  
JJ Sixma ◽  
PG de Groot
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Shear Rates ◽  
Wall Shear ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti- GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.

Platelet Deposition Induced by Severely Damaged Vessel Wall Is Inhibited by a Boroarginine Synthetic Peptide with Antithrombin Activity

1994 ◽  
Vol 71 (04) ◽  
pp. 511-516 ◽  
Author(s):  
J J Badimon ◽  
D Weng ◽  
J H Chesebro ◽  
V Fuster ◽  
L Badimon
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Synthetic Peptide ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Blood Platelet ◽  
Flow Conditions ◽  
Local Flow ◽  
Platelet Deposition ◽  
Damaged Vessel

SummaryThrombin plays a key role in platelet activation and thrombosis. Specific inhibition of thrombin appears to be one of the best approaches to prevent thrombus formation. We have studied the effects of a synthetic a-aminoboronic acid derivative - [Ac, (D) Phe-Pro-Boro-Arg-Hydrocloric acid] - on platelet deposition on severely damaged arterial wall. Platelet deposition was evaluated under well characterized rheological conditions in an original perfusion chamber and detected by autologous mIn-labeled platelets. The study was performed “in vivo” in a porcine model of arterial thrombosis triggered by severely damaged vessel wall at blood flow conditions mimicking mild stenosis (1690 s−1) and patent (212 s−1) vessels. In addition, ex-vivo platelet aggregation activity was evaluated by whole blood impedance aggregometry using collagen, ADP and thrombin as agonists. The synthetic a-aminoboronic peptide was intravenously administered as a bolus followed by continuous infusion. Ex vivo thrombin-induced whole blood platelet aggregation was totally abolished, while ADP- and Collagen-induced whole blood platelet aggregation was not modified. The effects of the synthetic antithrombin on platelet deposition were evaluated in native blood (non-anticoagulated) conditions and in combination with heparin. Under both experimental conditions, the synthetic peptide significantly inhibited platelet deposition at local flow conditions of both high (1690 s−1) and low (212s−1) shear rates. Our results suggest that specific inhibition of locally generated thrombin might be a good strategy to prevent platelet dependent arterial thrombus formation independently of the local flow shear rate of the area at risk.

Upstream Thrombus Growth Impairs Downstream Thrombogenesis in Non-Anticoagulated Blood: Effect of Procoagulant Artery Subendothelium and Non-Procoagulant Collagen

1991 ◽  
Vol 65 (05) ◽  
pp. 596-600 ◽  
Author(s):  
Kjell S Sakariassen ◽  
Harvey J Weiss ◽  
Hans R Baumgartner
Keyword(s):  
Shear Rate ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Rabbit Aorta ◽  
Axial Position ◽  
Fibrillar Collagen ◽  
Fibrin Deposition ◽  
Clotting Factors ◽  
Shear Rates ◽  
Platelet Deposition

SummaryIn the present experiments we have investigated the influence of wall shear rate and axial position on platelet and fibrin deposition which results when flowing human non-anticoagulated blood is exposed to either non-procoagulant fibrillar collagen (human type III) or procoagulant subendothelium (rabbit aorta). Platelet adhesion, thrombus volume and fibrin deposition were morphometrically evaluated at axial positions of 1 and 13 mm following perfusions for 5 min at shear rates of 100, 650 and 2,600 s-1.An axially-dependent decrease of platelet adhesion (34-57%, p <0.01-0.05) and thrombus volume (57-80%, p <0.05) was observed on collagen at all shear rates. On subendothelium, an axially-dependent decrease was observed for platelet adhesion only at 100 s-1 (29% ; p <0.01) and for thrombus volume at shear rates of 650 s-1 and above (49-58%, p <0.01). Deposition of fibrin on subendothelium was axially decreased (16-42%, p <0.05) at all shear rates, while no significant axial differences were seen on collagen. However, substantially more fibrin was deposited on the subendothelium (p <0.05), and the upstream platelet adhesion and thrombus volume were lower than on collagen (p <0.05) at 100 s-1 and 650 s-1. The axially-dependent phenomena on the two surfaces are consistent with the concept of rapid-growing upstream thrombi which deplete the blood layer streaming adjacent to the surface of platelets, leading to decreased platelet deposition farther downstream. The observations suggest that deposition of fibrin is enhanced by subendothelial tissue factor, and that upstream depletion of clotting factors may lower the downstream deposition of fibrin, analogous to the depletion of platelets.

The Effect of Platelet PlA Polymorphism on Experimental Thrombus Formation in Man Depends on Blood Flow and Thrombogenic Substrate

2001 ◽  
Vol 85 (06) ◽  
pp. 1097-1103 ◽  
Author(s):  
Kjell Sakariassen ◽  
Hélène Grandjean ◽  
Claire Thalamas ◽  
Bernard Boneu ◽  
Pierre Sié ◽  
...  
Keyword(s):  
Blood Flow ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Flow Properties ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Platelet Receptor ◽  
Platelet Deposition ◽  
Coronary Syndromes ◽  
Platelet Thrombus

SummaryA number of studies have reported conflicting data on the association of the PlA1/PlA2 polymorphism of the GPIIIa gene and coronary syndromes. We have investigated the effect of this polymorphism on experimental platelet thrombus formation in man. Forty healthy male volunteers were genotyped for the PlA1/PlA2 polymorphism. Thrombus formation was induced ex vivo by exposing a tissue factor (TF) or a collagencoated coverslip in a parallel plate perfusion chamber to native blood for 2 and 4 min. The shear rates at these surfaces were 650 and 2,600 s–1. Platelet and fibrin deposition was quantified by immunoenzymatic methods. The frequencies of PlA1/PlA1 and PlA1/PlA2 genotypes were 52.5% and 47.5%, respectively. Ex vivo deposition of fibrin on TF was not affected by the PlA1/PlA2 polymorphism. However, the ex vivo platelet deposition at 650 s–1 was higher in blood from PlA1/PlA1 individuals than in PlA1/PlA2 individuals (P = 0.008 at 4 min). On collagen, neither fibrin nor platelet deposition was significantly affected by the PlA1/PlA2 polymorphism. Platelet thrombus formation is significantly influenced by genetic variations in the GPIIIa platelet receptor. This effect depends on the blood flow properties and the nature of the thrombogenic stimulus.

The contribution of thrombin-induced platelet activation to thrombus growth is diminished under pathological blood shear conditions

2012 ◽  
Vol 107 (02) ◽  
pp. 328-337 ◽  
Author(s):  
Hannah Lee ◽  
Sharelle Sturgeon ◽  
Shaun Jackson ◽  
Justin Hamilton
Keyword(s):  
Platelet Activation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Direct Result ◽  
Protease Activated Receptors ◽  
Heart Attacks ◽  
Shear Rates ◽  
Coronary Syndromes

SummaryDeveloping novel anti-platelet therapies is an important clinical strategy for the prevention of arterial thromboses which cause heart attacks and most strokes. Thrombin activates platelets via protease-activated receptors (PARs), and PAR antagonists are currently under investigation as antithrombotics. Yet despite these clinical advances, the importance of PARs to platelet activation during thromboses formed under pathological conditions has not been investigated. To this end, we examined the role of PAR-dependent platelet activation in thrombus formation in the presence of elevated blood shear rates. We used two in vivo thrombosis models and an ex vivo whole blood flow approach in PAR4-/-mice, whose platelets are unresponsive to thrombin, to show that the contribution of PAR-mediated platelet activation to thrombosis is diminished at pathological blood shear rates as a direct result of decreased incorporation of thrombin-activated platelets into growing thrombi. Our ex vivo observations were replicated in human whole blood treated with a PAR1 antagonist. These results define a novel, shear-regulated role for thrombin/PAR-dependent platelet activation during thrombosis and provide important insights into the conditions under which PAR antagonists may best be used for the prevention of acute coronary syndromes.

Sub-Optimal Inhibition of Thrombus Formation by Aspirin (as measured by RTTP analysis) In Primary Thrombocythemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4218-4218
Author(s):  
Gillian Stephens ◽  
Jan Tauscher ◽  
Fabian Siegel ◽  
David R. Phillips ◽  
Patrick Andre ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Real Time ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Myeloproliferative Disease ◽  
Fluorescent Intensity ◽  
Shear Rates ◽  
Primary Thrombocythemia ◽  
Platelet Deposition

Abstract Abstract 4218 Primary thrombocythemia (PT) is a myeloproliferative disease with a high incidence of thrombotic complications. First-line therapy is the inhibition of platelet aggregation with aspirin (via COX-1 inhibition) for the prevention of thromboembolic complications. However, this does not account for the potential for COX-2 expression in newly synthesized platelets. The purpose of the present study was 1) to determine whether aspirin reduces the thrombotic potential of PT patients who did not undergo cytoreductive therapy to the levels of those achieved in healthy volunteers (HV), and 2) to determine whether the presence of the JAK2V617F mutation is responsible for the elevated thrombotic potential in PT patients. We determined the thrombotic potential of a cohort of 16 PT patients and compared these values to those of 10 healthy volunteers all treated with 100mg aspirin QD. Thrombotic potential was determined using a custom built Real Time Thrombosis Profiler (RTTP). Whole blood anticoagulated with a Factor Xa inhibitor (to preserve physiological Ca++ concentration) with Rhodamine 6G-labeled platelets was perfused over a fibrillar collagen surface at shear rates approximating those in veins (100s-1), arteries (600 s-1) and moderately stenosed arteries (1600s-1). The deposition of fluorescently labeled platelets on the collagen surface was monitored in real time by fluorescence microscopy in the RTTP. Endpoint thrombosis (size of thrombi at t=300sec expressed as Mean Fluorescent Intensity/Area of coverage) and rate of thrombus growth (initial rate of platelet deposition) were recorded for each individual as measures of thrombotic potential and data are expressed as mean ± SEM (statistics performed using GraphPad Prism v4.03 using unpaired, 2-tailed Students t-test). Despite aspirin therapy, PT patients were characterized by significant increases in thrombotic potential at venous and arterial shear rates (Table 1) with a greater than 2-fold increase in the rate of platelet deposition. Interestingly only the size of thrombi formed but not the rate of formation was elevated in PT patients at moderately stenosed shear, likely a reflection of the increasing contribution of shear and decreasing dependence on absolute platelet count and other individual cellular factors in whole blood. The Disclosures: No relevant conflicts of interest to declare.

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