Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry

Abstract Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light- scatter profile. Alternatively, platelets could be identified with FITC- AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin- PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose- dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.

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Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry

Blood ◽

10.1182/blood.v70.1.307.bloodjournal701307 ◽

1987 ◽

Vol 70 (1) ◽

pp. 307-315 ◽

Cited By ~ 3

Author(s):

SJ Shattil ◽

M Cunningham ◽

JA Hoxie

Keyword(s):

Flow Cytometry ◽

Monoclonal Antibodies ◽

Whole Blood ◽

Thrombus Formation ◽

Fluorescein Isothiocyanate ◽

Platelet Surface ◽

Fibrinogen Receptor ◽

Activated Platelets ◽

Clinical Disorders ◽

Granule Membrane

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light- scatter profile. Alternatively, platelets could be identified with FITC- AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin- PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose- dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.

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DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

10.1055/s-0038-1643830 ◽

1987 ◽

Cited By ~ 4

Author(s):

S J Shattil ◽

J A Hoxie ◽

M Cunningham ◽

C S Abrahms ◽

J O’Brien ◽

...

Keyword(s):

Flow Cytometry ◽

Monoclonal Antibodies ◽

Platelet Activation ◽

Whole Blood ◽

Thrombus Formation ◽

Membrane Surface ◽

White Blood Cells ◽

Color Flow ◽

Platelet Surface ◽

Activated Platelets

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

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Platelet Hyporeactivity in Very Low Birth Weight Neonates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656093 ◽

1997 ◽

Vol 77 (05) ◽

pp. 1002-1007 ◽

Cited By ~ 84

Author(s):

Damodara Rajasekhar ◽

Marc R Barnard ◽

Francis J Bednarek ◽

Alan D Michelson

Keyword(s):

Flow Cytometry ◽

Binding Site ◽

Whole Blood ◽

Very Low Birth Weight ◽

Thromboxane A2 ◽

Platelet Surface ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Platelet Binding ◽

Thromboxane A2 Analogue

SummaryVery few studies have examined platelet function in very low birth weight (VLBW) preterm neonates, because of the relatively large volumes of blood required. In this study, platelet function in clinically stable VLBW neonates was examined by whole blood flow cytometry, which requires only 5 |jl1 of whole blood per assay. The following monoclonal antibodies were used: S12 (P-selectin-specific, reflecting a granule secretion), PAC1 (directed against the fibrinogen binding site exposed on the GPIIb-IIIa complex of activated platelets), F26 (directed against a conformational change in fibrinogen bound to the GPIIb-IIIa complex), and 6D1 (directed against the von Willebrand factor binding site on the GPIb-IX-V complex). VLBW neonates, like normal adults, did not have circulating activated platelets, as determined by the lack of binding of SI2, PAC1, and F26 in the absence of an added agonist. VLBW neonatal platelets were markedly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619 (a stable thromboxane A2 analogue), as determined by the extent of increase in the platelet binding of SI2, PAC1, and F26, and the extent of decrease in the platelet binding of 6D1. In summary, compared to adults, the platelets of VLBW neonates are markedly hyporeactive to thrombin, ADP/epinephrine and a thromboxane A2 analogue in the physiologic milieu of whole blood, as determined by: 1) the increase in platelet surface P-selectin; 2) the exposure of the fibrinogen binding site on the GPIIb-IIIa complex; 3) fibrinogen binding; and 4) the decrease in platelet surface GPIb. This platelet hyporeactivity may be a factor in the propensity of VLBW neonates to intraventricular hemorrhage. In addition to its previously defined use as a test of platelet hyperreactivity, the present study suggests that whole blood flow cytometry may be useful in the clinical assessment of platelet hyporeactivity.

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Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry

Blood ◽

10.1182/blood.v69.5.1401.bloodjournal6951401 ◽

1987 ◽

Vol 69 (5) ◽

pp. 1401-1403

Author(s):

GI Johnston ◽

EB Pickett ◽

RP McEver ◽

JN George

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Response ◽

Platelet Surface ◽

Activated Platelets ◽

Low Concentrations ◽

Granule Membrane ◽

Membrane Changes ◽

Granule Secretion

Platelet membrane changes that accompany in vivo activation may be difficult to detect if only a small fraction of circulating platelets has undergone secretion. This study describes an approach to that problem by using a method to measure the number of molecules of fluorescein-labeled antibody bound to individual platelets by flow cytometry. The platelet response to different concentrations of thrombin was determined by measuring the binding of a monoclonal antibody (S12) to GMP-140, an alpha-granule membrane protein that becomes exposed on the platelet surface during alpha-granule secretion. Unstimulated platelets bound a mean of 1,120 molecules of S12 per cell, and 93% of platelets bound less than 2,000 molecules. Platelet stimulation by 0.25 U/mL thrombin caused maximum S12 binding with a mean of 7,529 molecules per cell. Even at low concentrations of thrombin (0.025 U/mL), 5% of platelets were maximally activated, binding over 7,000 molecules of S12 per cell. Conversely, at 0.25 U/mL thrombin, 13% of platelets continued to bind less than 2,000 molecules of S12 per cell. A mixture of as little as 5% thrombin-activated platelets with unstimulated platelets could be detected by this method. Therefore flow cytometry offers an important tool for investigating patients who may have circulating activated platelets as part of a disorder predisposing to thrombosis or hemorrhage.

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Comparison of Platelet Function and Bleeding in Thrombocytopenic Patients with Immune Thrombocytopenic Purpura (ITP) and Chemotherapy-Induced Thrombocytopenia (CIT).

Blood ◽

10.1182/blood.v110.11.2094.2094 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2094-2094 ◽

Cited By ~ 1

Author(s):

Bethan Psaila ◽

James B. Bussel ◽

Matthew D. Linden ◽

You Fu Li ◽

Marc R. Barnard ◽

...

Keyword(s):

Flow Cytometry ◽

Blood Flow ◽

Platelet Function ◽

Whole Blood ◽

Bleeding Risk ◽

Severe Thrombocytopenia ◽

Platelet Surface ◽

Platelet Counts ◽

Activated Platelets ◽

Immature Platelet Fraction

Abstract Because of methodologic problems, platelet function in thrombocytopenic patients has never been adequately studied, and therefore individual differences in bleeding risk are poorly understood. Whole blood flow cytometry analyzes the function of individual platelets, thereby enabling assessment of platelet function in severe thrombocytopenia (Michelson, A. Platelets, 2nd ed, Elsevier, 2007). In this study, platelet function was compared in 31 ITP patients, 14 CIT patients, and 16 healthy controls. Clinical bleeding was related to platelet function testing. The immature platelet fraction (IPF, or reticulated platelets) was measured in a Sysmex XE-2100. Platelet surface P-selectin and activated integrin αIIbβ3 (reported by monoclonal antibody PAC1) were measured by whole blood flow cytometry in the presence and absence of 0.5 μM ADP, 20 μM ADP, 1.5 μΜ TRAP, or 20 μM TRAP. Bleeding was quantified by a comprehensive score that allocates grades of 0 (no), 1 (minor) or 2 (marked) bleeding at 10 anatomic sites (Page, L.K. Br J Haematol 2007). Mean platelet volume (MPV) and IPF were higher in ITP than CIT, reflecting, as expected, a higher rate of platelet production (Table 1). Platelet surface P-selectin without added agonist (i.e. circulating activated platelets) was significantly higher in both ITP and CIT patients than in controls (Table 2). However, upregulation of platelet surface P-selectin and activated αIIbβ3 in response to ADP and TRAP (platelet ‘activatability’) was reduced in CIT patients compared with ITP patients and controls (Table 2). Stratification of bleeding scores by platelet count showed that CIT patients had more GI, urinary, and pulmonary bleeding at platelet counts <20 × 109/L whereas ITP patients had more skin/oral bleeding (Table 1). In sum, the higher platelet surface P-selectin and activated αIIbβ3 in CIT and ITP patients than in controls is consistent with a role for circulating activated platelets in maintenance of vascular integrity in thrombocytopenia. However, platelet activation in response to ADP and TRAP is reduced in CIT compared with both ITP and controls, which may reflect the relative senescence (as evidenced by lower IPF) of CIT platelets and/or effects of chemotherapy or the underlying leukemia. These data demonstrate that bleeding in different thrombocytopenic conditions is not entirely explained by the thrombocytopenia per se. Reduced responses to ADP and TRAP in CIT patients compared with ITP patients may be clinically significant, given that, at equivalent degrees of thrombocytopenia, CIT patients had more significant bleeding (GI, urinary, pulmonary) than ITP patients. Table 1. All Patients Patients with platelet counts <20 × 109/L MPV IPF% Absolute IPF ×109/L Skin/oral bleeding Bleeding other than skin/oral *P <0.05 CIT 7.2 11.0 1.7 6/10 5/10 ITP 9.2* 18.1 5.8* 10/11 0/11* Table 2. (mean fluorescence intensity) CIT ITP Controls a = P<0.05 for CIT vs ITP; b = P<0.05 for ITP or CIT vs controls No Agonist P-selectin 7.6b 6.2b 3.1 ActivatedαIIbβ3 7.0 10.2 7.2 High ADP P-selectin 41.8a,b 114.0 87.5 ActivatedαIIbβ3 154.4a,b 390.2 381.7 High TRAP P-selectin 69.9a,b 296.6 505.6 ActivatedαIIbβ3 46.9a,b 241.2b 457.7

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A Flow Cytometry Method for Characterizing Platelet Activation

2020 Design of Medical Devices Conference ◽

10.1115/dmd2020-9070 ◽

2020 ◽

Author(s):

Brian Alzua ◽

Mark Smith ◽

Yan Chen

Keyword(s):

Flow Cytometry ◽

Medical Device ◽

Platelet Activation ◽

Medical Devices ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Standard Regimen ◽

Activated Platelets ◽

Wide Range

Abstract Hemocompatibility testing is critical for assessing the safety of blood-contacting medical devices. Comprehensive hemocompatibility testing requires examining a wide range of possible adverse effects cause by direct or indirect blood contact, such as hemolysis, complement activation, and thrombus formation [1]. Moreover, these domains each encompass complex intercellular processes with many potential targets for analysis. For example, the current testing paradigm of platelet function may involve exposing the device to human whole blood and performing simple blood counts and/or macroscopic evaluation to determine the extent of platelet activation and clot formation as described in ASTM F2888-19. However, this approach does not capture any observations for device-mediated initiation of any steps in the platelet activation pathway prior to aggregation. We have validated a method to evaluate platelet activation by quantifying surface p-selectin expression after exposure to various materials. This method will provide an additional level of detail about potential platelet activating properties of a medical device. Flow cytometry has been used previously to measure platelet activation for clinical and research purposes. We sought to adapt this method to test for platelet activation induced by exposure of blood to medical devices or materials. We determined that processing fresh whole blood to platelet-rich plasma (PRP) by gentle centrifugation enhanced the signal compared to fresh blood itself. In each experiment, devices were exposed to PRP according to an extraction ratio of 6 cm2/mL for 1 hour. A blank control consisting of untreated PRP, and a positive control consisting of ADP, a potent agonist, were also used. After the exposure, excess plasma was removed from the articles and combined with anti-CD61 (to stain for platelets) and anti-CD62P (to stain for activated platelets) antibodies. Flow cytometry was then performed to quantify the percentage of CD62P+ over the total CD61+ cells to measure the percentage of activated platelets. In order to optimize the method, we investigated the effect of several experimental factors, including anticoagulant usage, donor variability, and selection of reference materials to serve as controls. Our results indicate that the flow cytometry-based method is consistent and reproducible, quick and easy to perform, and is well-correlated with results from the standard platelet and leukocyte count assay. The flow cytometry-based platelet activation method is a powerful supplement to the standard regimen of medical device hemocompatibility testing.

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Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry

Blood ◽

10.1182/blood.v69.5.1401.1401 ◽

1987 ◽

Vol 69 (5) ◽

pp. 1401-1403 ◽

Cited By ~ 54

Author(s):

GI Johnston ◽

EB Pickett ◽

RP McEver ◽

JN George

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Response ◽

Platelet Surface ◽

Activated Platelets ◽

Low Concentrations ◽

Granule Membrane ◽

Membrane Changes ◽

Granule Secretion

Abstract Platelet membrane changes that accompany in vivo activation may be difficult to detect if only a small fraction of circulating platelets has undergone secretion. This study describes an approach to that problem by using a method to measure the number of molecules of fluorescein-labeled antibody bound to individual platelets by flow cytometry. The platelet response to different concentrations of thrombin was determined by measuring the binding of a monoclonal antibody (S12) to GMP-140, an alpha-granule membrane protein that becomes exposed on the platelet surface during alpha-granule secretion. Unstimulated platelets bound a mean of 1,120 molecules of S12 per cell, and 93% of platelets bound less than 2,000 molecules. Platelet stimulation by 0.25 U/mL thrombin caused maximum S12 binding with a mean of 7,529 molecules per cell. Even at low concentrations of thrombin (0.025 U/mL), 5% of platelets were maximally activated, binding over 7,000 molecules of S12 per cell. Conversely, at 0.25 U/mL thrombin, 13% of platelets continued to bind less than 2,000 molecules of S12 per cell. A mixture of as little as 5% thrombin-activated platelets with unstimulated platelets could be detected by this method. Therefore flow cytometry offers an important tool for investigating patients who may have circulating activated platelets as part of a disorder predisposing to thrombosis or hemorrhage.

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Effects of leukocyte-derived cathepsin G on platelet membrane glycoprotein Ib-IX and IIb-IIIa complexes: a comparison with thrombin

Blood ◽

10.1182/blood.v82.8.2442.bloodjournal8282442 ◽

1993 ◽

Vol 82 (8) ◽

pp. 2442-2451

Author(s):

M Molino ◽

M Di Lallo ◽

N Martelli ◽

G de Gaetano ◽

C Cerletti

Keyword(s):

Polymorphonuclear Leukocytes ◽

Fluorescein Isothiocyanate ◽

Adenosine Diphosphate ◽

Cathepsin G ◽

Platelet Surface ◽

Fibrinogen Receptor ◽

Antigenic Sites ◽

Triton X ◽

Von Willebrand ◽

Willebrand Factor

Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC- 1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA- fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb- IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.

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Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽

10.1182/blood.v83.4.1006.1006 ◽

1994 ◽

Vol 83 (4) ◽

pp. 1006-1016 ◽

Cited By ~ 29

Author(s):

AD Cox ◽

DV Devine

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Activation ◽

Factor Xiiia ◽

Cross Linking ◽

Platelet Surface ◽

Activated Platelets ◽

Fibrinogen Binding ◽

A Chain ◽

Functional Receptor

Abstract Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

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