Increase In Platelet Concentration Research Articles

Growth factor and cytokine characterization of canine platelet lysate with variable leukocyte concentration, plasma content, and heat-sensitive proteins.

Platelet lysate is an acellular platelet product containing factors released from secretory granules, including cytokines and growth factors. This study aimed to evaluate different centrifugation methods used to prepare canine platelet lysate with variable content of leukocytes, plasma, and heat-sensitive proteins. Whole blood was collected from six dogs and two double-spin preparation methods were used to generate the platelet-rich plasma with reduced (PRP) and high (L-PRP) concentration of leukocytes. A portion of both methods underwent plasma depletion via centrifugation and platelet lysate was generated via freeze-thaw cycles. A portion of the generated platelet lysate underwent complement inactivation via heat treatment. Growth factors (TGF-β1, VEGF, TNF-α, PDGF-BB, HGF) were quantified in all different platelet lysate preparations using ELISAs. Both platelet-rich plasma preparations had a 6.7-fold increase in platelet concentration. White blood cell (WBC) concentration compared to whole blood increased 1.2-fold times in PRP and 1.9-fold times in L-PRP. Negligible concentrations of platelets, WBC, and hematocrit were identified in all lysate groups. Statistically significant differences were identified for PDGF, VEGF, and TNF-α, and not for TGF-β or HGF. No growth factor differences were noted between centrifugation methods. PDGF was significantly higher in platelet lysate that was plasma depleted. VEGF was significantly higher in heat-treated lysate groups. TNF-α concentrations were overall very low, though were noted to significantly increase following plasma depletion. These results support that growth factors and cytokine release can be affected by the platelet lysate preparation and processing.

Application of Platelet-Rich Gel in Repairing Scalp Defects Combined with Skull Exposure: A Case Series Analysis

Abstract Clinical treatment of exposed skull injuries becomes extremely challenging. The current treatment methods for exposed skull injuries are mainly surgical treatments. However, these treatments are not suitable for elderly patients who cannot tolerate anaesthesia. This research aimed to assess the effectiveness of platelet-rich gel (PRG) as a non-surgical option for repairing exposed skull injuries. In this study, twelve patients who presented to our hospital with scalp defects between September 2018 and March 2022 were included. Patients were evaluated before and three days after treatment using the Visual Analog Scale (VAS) score, inflammatory markers, and topical skin temperature. Additionally, the length of the platelet-rich gel, number of dressing changes, wound-healing time, local scarring, and hair growth were recorded. The average wound area was 17.08 SD5.49 cm2. The platelet-rich gel exhibited a length of 3.09 SD0.09 cm and showed a significant increase in platelet concentration by a factor of 2.43 SD0.07. all 12 patients achieved complete healing, requiring average number of 8.3 SD2.0 dressing changes and resulting in an average wound healing time of 25.0 SD5.8 days. There were no statistically significant differences in the Visual Analog Scale scores, inflammatory markers, or topical skin temperatures before and after treatment. Follow-up assessments conducted 4–10 months after treatment revealed minimal local scars and sparse hair growth. The one-step preparation of platelet-rich gel in reconstructing scalp defects is a simple, safe, and effective non-surgical method. Its advantages include speedy preparation, painless treatment, and small local scars.

Comparison of Resuspension Techniques on the Cellular and Cytokine Characteristics of Platelet Rich Plasma: A Pilot Study

Background: Platelet Rich Plasma (PRP) is increasingly used for a variety of musculoskeletal ailments such as tendinopathy and arthritis. There are numerous protocols for preparing PRP, but to our knowledge there have been no studies to assess the impact that re-suspension techniques may have on the cellular characteristics and growth factor expression of the final PRP product. Hypothesis: The type of re-suspension technique used will alter the platelet characteristics and growth factor expression of PRP. Study Design: Descriptive laboratory study. Methods: Blood was collected from nine healthy volunteer donors between ages 26-60. Informed consent and ethics approval was obtained. A double spin technique was used resulting in a concentrated PRP product. Three commonly employed re-suspension techniques were utilized to re-suspend the final PRP product and the cellular characteristics and growth factor expression were analyzed using a reproducible protocol. This study was completed over a single day. Results: All three re-suspension techniques resulted in a 4.6-5.2-fold increase in platelet concentration with no difference between groups (p=0.31). This is consistent with the recommended PRP concentration in published literature. Each re-suspension technique also demonstrated in increase in concentration of PDGF and VEGF with no difference between groups (p=0.81 and p=0.97). Conclusion: This pilot study suggests that the re-suspension techniques may be equivalent and do not result in altered cellular or growth factor characteristics within the study limitations. Clinical Relevance: The mixing method can be labor and time intensive and prolong patient wait times. The vortex method can shorten the overall patient encounter time while still providing an optimal PRP product. Previously, there were only theoretical assumptions regarding how the re-suspension techniques may modify PRP characteristics.

The Influence of Platelet Concentrate on the Development of Cattle Embryos in аn In Vitro System

There are many studies on the improvement of the conditions for cultivating bovine embryos in vitro. However, the issue of co-cultivation of platelets and cow embryos is understudied, although platelet activation leads to the release of a considerable amount of biologically active substances into the culture medium and their co-cultivation with embryos can positively affect the growth and development of the latter. Therefore, the purpose of this study was to investigate the effect of different platelet concentrations in the in vitro culture system on the development of cow embryos. Fertilized zygotes (total number 180) were divided into 5 groups: Group 1 – culture medium without platelet addition (control); Group 2 – medium for cultivation with the addition of 10×106 platelets/cm3 ; Group 3 – final concentration in the system is 20×106 platelets/cm3 ; Group 4 – 50×106 platelets/cm3 ; Group 5 – 100×106 platelets/cm3 . According to the results, it was found that co-cultivation of embryos with platelets in the in vitro system is effective. Thus, a correlation was found between improved embryo development indicators and an increase in platelet concentration. The optimal platelet concentration was 50×106 /cm3 , which allowed obtaining a 13.9% higher level of blastulation, 15.7% higher average embryo size, and 2.5% higher average number of cells in the blastocyst compared to the control. At the same time, the platelet concentration of 100×106 /cm3 led to a significant decrease in the indicators under study, compared with the group without platelets. Therefore, co-cultivation of cow embryos with platelets is advisable since it allows improving the development indicators of cow embryos. The data analysed and presented in this paper will increase the efficiency of cultivation of bovine embryos for both scientific and industrial purposes

Simple Tube Centrifugation Method for Platelet-Rich Plasma (PRP) Preparation in Catalonian Donkeys as a Treatment of Endometritis-Endometrosis.

Simple SummaryPlatelet-rich plasma (PRP) is used to improve the regenerative capacity of damaged tissues in different species. In equine medicine, PRP is commonly used to treat joint diseases, tendinitis, ligamentous lesions, and persistent endometritis. Jenny endometrium shows a high sensitivity to endometritis. There are important differences between donkey and horse blood characteristics. Several protocols to obtain horse PRP have been reported, but no protocols have yet been reported for obtaining donkey PRP. Our study shows that single-spin tube centrifugation at 133× g for 10 min is appropriate to obtain donkey PRP with therapeutic potential in jenny endometritis-endometriosis.The aim of this study was to standardize a simple, manual platelet-rich plasma (PRP) protocol in Catalonian donkeys using single-spin tube centrifugation as a treatment for jenny endometritis. The objective was to obtain a blood product with a moderate concentration of platelets (2 or 3 times baseline physiologic values) and a low WBC (White Blood Cells) concentration. Blood was drawn from six Catalonian donkeys using acid citrate dextrose (ACD) as an anticoagulant, and then processed by single centrifugation at 133× g for two different centrifugation times (10 and 15 min). The PRP samples were evaluated by flow cytometry, and TGF-β1 (Transforming Growth Factor-Beta1) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). The 10 min centrifugation protocol resulted in a slightly greater release of TGF-β1 (6044.79 ng/mL), a 2.06-fold increase in platelet concentration, and a 15-fold reduction in leukocyte concentration when compared to the initial values. The 15 min centrifugation time resulted in a 2.44-fold increase in baseline platelet concentration, a reduction in WBC count by a factor of 20, and slightly lower TGF levels (5206 ng/mL). We conclude that both protocols are adequate for the obtention of PRP, and both may have an acceptable therapeutic potential for use in this species, although this needs to be further validated.

484 FDA cleared devices produce inconsistent platelet-rich plasma product

Platelet-rich plasma (PRP) has emerged as a novel option to treat androgenetic alopecia. However, data focused on quality of product from FDA cleared devices is lacking. Herein, we assessed and compared cellular PRP content produced from FDA cleared devices. Within one academic institution, white blood cell (WBC) counts and platelet counts from whole blood and resultant PRP were retrospectively reviewed. Institutional quality control (QC) procedures were utilized to obtain counts. Of the fifty-two PRP samples assessed, only 2 met the Insitutions QC standard (defined a 3-fold increase in platelet concentration). Paired analysis demonstrated similar platelet counts in whole blood and PRP. Platelet counts from individual devices were significantly different. Within the devices, platelet counts from PRP samples were inconsistent, failing the Shapiro-Wilk test. The Emcyte device demonstrated the highest capacity to concentrate, even though mean enrichment was just 2.9-fold and capture efficiency was found to be 34%. WBC numbers were negligible in Eclipse and Selphyl final PRP samples. However, the Emcyte device enriched PRP with WBCs similar to or greater than whole blood. These results demonstrate inconsistent PRP end product with variable platelet counts among multiple devices and within devices. QC testing standards were not passed by most samples. Efficiency of platelet capture was lower than that claimed by device manufacturers. WBC content varied by device used for preparation. Of note, no patients worsened while receiving these treatments. This study presents an additional variable in PRP preparation – inconsistent PRP product from FDA-cleared devices. Additionally, clinical improvement despite QC failure suggests platelet numbers may not be vital for clinical improvement.

Assessment of Canine Autologous Conditioned PlasmaTM Cellular and Transforming Growth Factor-β1 Content.

To evaluate (1) the cellular composition of canine ACP™ including using two different preparation protocols with variations on centrifugation time, (2) the effect of different activation protocols on the transforming growth factor (TGF)-β1 content in the ACP, and (3) patient factors that might influence platelet concentration of the ACP.ACP was made with blood from 15 dogs using a manufacturer-recommended protocol[1]. Each ACP sample was divided into three aliquots that were activated with calcium chloride (CaCl2), human γ-thrombin (HGT), or not activated. TGF-β1 was quantified in each aliquot using an ELISA and comparisons among activation protocols were performed using a Skillings-Mack test. Correlations between platelet and TGF-β1 concentration were assessed with a Pearson correlation coefficient. ACP was subsequently prepared from an additional 17 dogs using a slightly modified centrifugation protocol and cellular composition was assessed. Effects of dog age, body weight, and hematocrit were assessed for their potential impact on ACP platelet concentration using a multiple linear regression analysis.The mean increase in platelet concentration in the ACP above that in the whole blood was 1.2× (±std 0.62) and leukocyte concentration was a mean of 26% (0.37) that in the whole blood using the standard protocol. There was a significant (p < 0.01) effect of activation on TGF-β1 concentrations with mean concentrations of 4,538 (2,317), 14,948 (13,784), and 14,096 (15,210) pg/ml in aliquots that were not activated or were activated with thrombin or CaCl2 respectively. There were significant correlations between the platelet concentration and TGF-β1 concentration in aliquots that were activated with either thrombin (r = 0.66; p < 0.01) or CaCl2 (r = 0.86, p < 0.0001). The mean increase in platelet concentration was 1.4× (0.62) and the leukocyte concentration was 0.28× (0.13) that in whole blood using the modified ACP preparation protocol. Dog age, body weight, and hematocrit were not significant predictors of ACP platelet concentration.These data show that on average this preparation protocol produces a mildly platelet-concentrated, leukoreduced platelet-rich plasma. Intentional activation had a significant effect on TGF-β1 concentrations with use of both CaCl2 and thrombin resulting in higher TGF-β1 concentrations than that obtained in samples that are not activated.

Normal platelet function in platelet concentrates requires non-platelet cells: a comparative in vitro evaluation of leucocyte-rich (type 1a) and leucocyte-poor (type 3b) platelet concentrates

BackgroundTherapeutic success of platelet-rich plasma (PRP) may vary based on the composition and preparation method. The objective of this study was to evaluate the cellular components of platelet concentrates produced...

Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors

IntroductionPlatelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed.MethodsPRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure.ResultsOur method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only <0.3% from the initial red blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF.ConclusionsOur study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool from different donors with high reproducibility. These findings support the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes.

Pharmacodynamic stimulation of thrombogenesis by angiotensin (1–7) in recurrent ovarian cancer patients receiving gemcitabine and platinum-based chemotherapy

This randomized, double-blind, placebo-controlled Phase 2 study evaluated safety and efficacy of A(1-7) for reduction in Grade 3-4 thrombocytopenia in patients receiving myelosuppressive chemotherapy. Pharmacodynamic activity of A(1-7) in platelet production and retention of scheduled dose intensity were also determined. Thirty-four patients with ovarian, Fallopian tube, or peritoneal carcinoma receiving gemcitabine and carboplatin or cisplatin were evaluated. Patients were randomized to receive study drug subcutaneously at 100 mcg/kg (n = 11), 300 mcg/kg (n = 13), or placebo (n = 10) following chemotherapy for up to six cycles. Hematologic variables were obtained throughout each treatment cycle. There were no drug-related safety issues. There were no instances of Grade 4 thrombocytopenia in patients who received 100 mcg/kg treatment compared to 6 % of chemotherapy cycles for patients receiving placebo (p = 0.07). The maximal percentage increase in platelet concentration from baseline was higher for patients who received 100 mcg/kg A(1-7) compared to placebo (p = 0.02). This increase was accompanied by a reduction in the nadir absolute neutrophil count (p = 0.04). Relative dose intensity for the combination chemotherapy was higher for patients who received 100 mcg/kg A(1-7) compared to placebo (p = 0.04). There were no differences in outcomes for patients receiving 300 mcg/kg dose compared to placebo. A 100 mcg/kg dose of A(1-7) was shown to produce pharmacodynamic effects on peripheral blood platelet counts, preserve planned dose intensity, and reduce Grade 3-4 thrombocytopenia following gemcitabine and platinum chemotherapy. These findings are consistent with A(1-7)-induced stimulation of thrombogenesis in the bone marrow following marrow-toxic chemotherapy.

Treatment of Knee Joint Osteoarthritis with Autologous Platelet-Rich Plasma in Comparison with Hyaluronic Acid

This study aimed to find a simple, cost-effective, and time-efficient method for the preparation of platelet-rich plasma (PRP), so the acquired benefits will be readily available for multiple procedures in smaller outpatient clinics and to explore the safety and efficacy of the application of PRP in the treatment of degenerative lesions of articular cartilage of the knee. The study was designed as a prospective, cohort study with a control group. A total of 120 patients with Grade 1, 2, or 3 osteoarthritis according to the Kellgren and Lawrence grading scale were enrolled in the study. One group of patients was treated using three intra-articular applications of PRP, and the second group of patients was given three injections of hyaluronic acid. Outcome measures included the Western Ontario and McMaster Universities Osteoarthritis Index and the 11-point pain intensity Numeric Rating Scale. On average, a 4.5-fold increase in platelet concentration was obtained in the PRP group. No severe adverse events were observed. Statistically significantly better results in the Western Ontario and McMaster Universities Osteoarthritis Index and Numeric Rating Scale scores were recorded in a group of patients who received PRP injections after a 3- and 6-mo follow-up. Our preliminary findings support the application of autologous PRP as an effective and safe method in the treatment of the initial stages of knee osteoarthritis. Further studies are needed to confirm these results and to investigate the persistence of the beneficial effects observed.

Platelet-Rich Plasma Differs According to Preparation Method and Human Variability

Varying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma. Three different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated. The content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed. Single-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in platelet and cell numbers as well as levels of growth factors regardless of separation method.

Preparation of platelet-rich plasma from small volume of whole blood - A simplified approach

Platelet-rich plasma (PRP) is frequently used to enhance wound healing in numerous surgical applications. There are many methods of preparation of PRP. The present report describes a laboratory technique which is rapid, simple and inexpensive for preparing PRP from small volume of whole blood. Whole blood was drawn from 30 patients undergoing oral and maxillofacial surgery. PRP was prepared by the modified open system. Platelet counts for whole blood and PRP were determined using a cell counter. The data from 30 patients aged 20 to 33 years, with a mean of 24.4 (± 3.3) and male : female ratio of 1:1.1, were analysed. The platelet count in PRP was 3,157,667 ± 984,380 while that in whole blood was 268,667 (± 64,678) platelets /cubic ml, reflecting an 11-fold increase in platelet concentration. No effect of gender or ageing on platelet count was observed. This study demonstrates a simple, rapid and inexpensive technique that is useful in obtaining platelets concentrated approximately 11-fold.

The Effects of Platelet-Rich Plasma on Tendon and Ligament: Basic Science and Clinical Application

The introduction of platelet-rich plasma (PRP) as a possible adjunct to conservative and operative treatment has motivated significant research in the topic. Platelet growth factors within alpha granules are released upon activation. These growth factors regulate and enhance tendon healing, so an increase in platelet concentration at the site of injury would hypothetically increase the healing response. Laboratory and animal studies have shown encouraging results in the role and effect of PRP on tendon healing. Animal studies also show an increased strength of tendon repairs augmented with PRP. However, clinical trials have been less consistent. Some show an improvement in pain control and return to function, whereas others show no difference compared to controls. The purpose of this review was to describe the biology and clinical outcomes of PRP treatment of tendon and ligament injuries.

Calidad del plasma rico en plaquetas: estudio de la activación plaquetaria

Objective. Platelet Rich Plasma (PRP) is an autologous preparation currently used in oral and maxillofacial reconstructive surgery. Blood collection and preparation of platelet concentrates may lead to platelet activation and the premature loss of their granular load. In this study, we have analyzed the quality of the PRP obtained from a small volume of whole blood through a double centrifugation technique, so called tube method. Design. We obtained 50 ml of whole blood from 45 patients and centrifuged at 200 g for 10 minutes. The plasma and buffy-coat were collected and we then centrifuged at 700g for 15 minutes. The pellet was resuspended after discarding 2/3 of the plasma. The platelet concentration, platelet activation and the functional response to thrombin were analyzed in these samples. Results. By using this method of PRP preparation, we obtained a 364 ± 177% increase in platelet concentration in comparison with whole blood levels. Platelet activation, measured by flow cytometry analysis of CD62 expression, was of 2.7% in unprocessed blood and 3.6% in fresh PRP. This figure increased to 16% in PRP samples after overnight storage at room temperature. A percentage of 96% of platelets showed activation in PRP samples after thrombin stimulation. Conclusion. Our results show that platelets contained in PRP concentrates obtained by this method are not significantly activated. A good functional platelet reserve is preserved through the procedure, since platelets maintained a satisfactory response to thrombin after PRP preparation.

Platelet Quantification and Growth Factor Analysis from Platelet-Rich Plasma: Implications for Wound Healing

Growth factors released from activated platelets initiate and modulate wound healing in both soft and hard tissues. A recent strategy to promote the wound-healing cascade is to prepare an autologous platelet concentrate suspended in plasma, also known as platelet-rich plasma, that contains growth factors and administer it to wound sites. The purpose of this study was to quantitate platelet number and growth factors released from a prepared platelet concentrate. Whole blood was drawn from 10 healthy patients undergoing cosmetic surgery and concentrated into platelet-rich plasma. Platelet counts on whole blood and platelet-rich plasma were determined using a Cell-Dyn 3200. Platelet-derived growth factor-BB, transforming growth factor-beta1, vascular endothelial growth factor, endothelial growth factor, and insulin-like growth factor-1 were measured in the platelet-rich plasma using the enzyme-linked immunosorbent assay method. In addition, platelet activation during the concentration procedure was analyzed by measuring P selectin values in blood serum. An 8-fold increase in platelet concentration was found in the platelet-rich plasma compared with that of whole blood (baseline whole blood, 197 +/- 42 x 10 platelets/microl; platelet concentrate, 1600 +/- 330 x 10 platelets/microl). The concentration of growth factors also increased with increasing platelet number. However, growth factor concentration varied from patient to patient. On average for the whole blood as compared with platelet-rich plasma, the platelet-derived growth factor-BB concentration increased from 3.3 +/- 0.9 ng/ml to 17 +/- 8 ng/ml, transforming growth factor-beta1 concentration increased from 35 +/- 8 ng/ml to 120 +/- 42 ng/ml, vascular endothelial growth factor concentration increased from 155 +/- 110 pg/ml to 955 +/- 1030 pg/ml, and endothelial growth factor concentration increased from 129 +/- 61 pg/ml to 470 +/- 320 pg/ml. No increase was found for insulin-like growth factor-1. In addition, no increase in platelet activation occurred during the concentration procedure as determined by the platelet surface receptor P selectin (45 +/- 16 pg/ml to 52 +/- 11 pg/ml, p = 0.65). In conclusion, a variety of potentially therapeutic growth factors were detected and released from the platelets in significant levels in platelet-rich plasma preparations. Sufficient concentrates and release of these growth factors through autologous platelet gels may be capable of expediting wound healing in a variety of as yet undetermined specific wound applications.

Platelet-rich plasma: harvesting with a single-spin centrifuge.

This paper discusses the results recently seen by practitioners who use platelet-rich plasma, as well as the practical considerations that they must make when considering whether to adapt their practice for its use. In this study, an average of a 358% increase in platelet concentration was achieved.

Platelets and Phospholipids in Tissue Factor-initiated Thrombin Generation

The influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 microM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 microM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 x 10(8)/ml to 0.5 x 10(8)/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient's blood was accelerated from approximately 10 min to 5 min when the platelet concentration increased from 0.05 x 10(8)/ml to 0.11 x 10(8)/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 x 10(8)/ml (range 0.5-3.1 x 10(8)/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 x 10(8)/ml to 3.1 x 10(8)/ml (4.0+/-0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 x 10(8)/ml.