Prolonged platelet hyperactivity after COVID-19 infection.

Platelet hyperactivity often occurs in patients with coronavirus disease 2019 (COVID-19). However, it remains unclear how long platelet hyperactivity lasts after the acute phase, owing to a lack of follow-up studies. To elucidate the course of platelet hyperactivity, we serially measured platelet activity in patients with COVID-19 up to 40 days after hospital admission using an easily assessable haematology analyser that semi-quantitates platelet clumps on a scattergram. Our results showed that platelet hyperactivity persisted for at least 40 days even after acute inflammation subsided in most patients with COVID-19, regardless of disease severity. Persistent platelet hyperactivity may contribute to thromboembolic complications in post-COVID-19 patients.

Influence of PAR-1 in patients with non-valvular atrial fibrillation: The antiplatelet effect of dabigatran.

INTRODUCTION: Dabigatran, a direct thrombin inhibitor, has been widely used in patients with non-valvular atrial fibrillation (NVAF) and is considered to have an antiplatelet effect. However, the mechanisms remain unclear. We evaluated protease-activated receptor-1 (PAR-1) expression and activation by thrombin on platelets from NVAF patients, before and after dabigatran treatment, in addition to the expression of platelet activation marker CD62P. MATERIALS AND METHODS: The study included 18 NVAF patients. We used flow cytometry to measure the binding of PAR-1 monoclonal antibodies (SPAN12 and WEDE15) and the expression of CD62P with and without thrombin stimulation, before, 14 days after, and 28 days after treatment with dabigatran. Coagulation fibrinolysis markers were also measured. RESULTS: PAR-1 expression was significantly lower in NVAF patients than in healthy controls (HC); it was further reduced by thrombin stimulation. CD62P expression was almost absent on the platelets in NVAF patients, but was significantly increased by thrombin stimulation. PAR-1 expression was not significantly different before and after treatment; CD62P expression was inhibited by dabigatran. The levels of coagulation markers were significantly higher in NVAF patients than in HC, and decreased after treatment. CONCLUSIONS: Lower expression of PAR-1 in NVAF patients resulted from the cleavage of PAR-1 on some platelets, by exposure to small amounts of thrombin in vivo. The therapeutic effect of dabigatran in NVAF patients was demonstrated by inhibition of CD62P expression on the platelet upon thrombin stimulation in vitro. Our results indicate that dabigatran may reveal antithrombotic activity with antiplatelet and anticoagulant effects.

Inhibitory Effects of P2Y12 Receptor Antagonist on PAR1- and PAR4-AP-Induced Platelet Aggregation in Patients with Stroke or TIA.

OBJECTIVES: The inhibitory effects of P2Y12 receptor antagonist on PAR1- and PAR4-activating peptide (AP)-induced platelet aggregation have not been fully elucidated. The present study aimed to investigate the inhibitory effects of P2Y12 receptor antagonist on PAR1- and PAR4-AP-induced platelet aggregation using platelet-rich plasma (PRP) from individuals including patients with stroke or transient ischemic attack (TIA). MATERIALS AND METHODS: PRP was given to 10 healthy individuals pretreated in vitro with cangrelor, then stimulated with adenosine diphosphate (ADP), PAR4-AP, or PAR1-AP. Moreover, 20 patients were enrolled from 148 consecutive patients with acute ischemic stroke or TIA admitted to our institute between December 2017 and April 2019. PRP obtained from each patient before and >7 days after initiation of clopidogrel was similarly stimulated with these agonists. Platelet aggregation was measured using an automatic coagulation analyzer in all participants. RESULTS: In healthy individuals, ADP- and PAR4-AP-induced platelet aggregations were significantly inhibited depending on the cangrelor concentration in vitro, while PAR1-AP-induced platelet aggregation was slightly inhibited. In patients with stroke or TIA, clopidogrel inhibited ADP-induced platelet aggregation at all concentrations, and significantly inhibited PAR4-AP-induced platelet aggregation at 50 µmol/L of PAR4-AP (p<0.05), especially in 5 patients who showed high reactivity to PAR4-AP. PAR1-AP-induced platelet aggregation was also slightly inhibited. CONCLUSIONS: We showed significant inhibitory effects on PAR4-AP-induced platelet aggregation by clopidogrel in patients with stroke or TIA who had high reactivity to PAR4-AP.

Thrombin-induced platelet aggregation -effect of dabigatran using automated platelet aggregometry.

Dabigatran, a direct oral thrombin inhibitor, has two therapeutic effects: anticoagulation; and antiplatelet activity. In the clinical field, evaluation of the effect of dabigatran on thrombin-induced platelet aggregation is difficult because of fibrin clot formation and platelet aggregation. The aim of this study was to establish a new platelet aggregation method and to investigate the effects of dabigatran on thrombin-induced platelet aggregation. Platelet aggregation with thrombin was performed with automated light transmission aggregometry (CS2400; Sysmex, Kobe, Japan) in 40 healthy subjects. Thrombin-induced platelet aggregation was performed using thrombin and platelet-rich plasma (PRP), and thrombin-induced fibrin polymerization was inhibited by adding the peptide Gly-Pro-Arg-Pro (GPRP). The effect of dabigatran was then evaluated using the above method. Thrombin at < 0.2 U/mL did not induce platelet aggregation in most normal subjects. Median maximum aggregation percent (MA%) (25th-75th percentile) with 0.5 and 1.0 U/mL of thrombin was 87.0% (79.3-90.8%), and 90.2% (86.5-92.2%), respectively. The anti-platelet effects of dabigatran were then evaluated with these concentrations of thrombin. Dabigatran (final concentration, 2.5-1000 nM) inhibited platelet aggregation by 0.2-1.0 U/mL of thrombin in a concentration-dependent manner in vitro. Dabigatran showed potent inhibitory effects against platelet aggregation induced by 0.5 and 1.0 U/mL thrombin with half maximal inhibitory concentrations of 10.5 and 40.4 nM, respectively. A standard for thrombin-induced platelet aggregation was developed using the CS2400 in healthy subjects, and dabigatran was confirmed to inhibit thrombin-induced platelet aggregation in vitro with PRP.

Pitfall of Light Transmission Aggregometry-Based Assessment of Platelet Function in Acute Ischemic Stroke Patients.

BACKGROUND: The utility of light transmission aggregometry (LTA)-based assessment of platelet function in acute ischemic stroke patients remains controversial. This study aimed to clarify why LTA failed to estimate platelet function in acute ischemic stroke patients. METHODS: Using LTA, we evaluated the platelet aggregation abilities of citrated blood samples from 22 acute noncardiogenic ischemic stroke patients prior to treatment and compared them with those of 65 heathy volunteer controls. Platelet counts and mean platelet volumes (MPV) of citrated blood and platelet-rich plasma (PRP) prepared for LTA were evaluated simultaneously. Using a hematology analyzer, we also measured and compared the aggregation-prone properties of platelets in the hematology analysis process between patient and control samples. RESULTS: Although platelets aggregated more easily and frequently in patient samples (P < .01), the maximum aggregation rate (MA%) of LTA was paradoxically lower in patients than in controls (P < .05). The PRP/citrated blood ratio of platelet counts and MPV were significantly lower in patients than in controls (P < .05). CONCLUSIONS: Our results suggest that MA% of LTA is erroneously displayed as lower values than the actual status in patients with increased platelet aggregation ability such as acute ischemic stroke because activated large platelets are preaggregated and thus decreased in the PRP on LTA.

Influence of Platelet Aggregate Formation in Blood Samples on Light Transmission Aggregometry Results.

BACKGROUND: Light transmission aggregometry is a standard method used to evaluate platelet function. However, in clinical settings, light transmission aggregometry results sometimes fail to reflect actual platelet hyperactivity. In patients with suspected platelet hyperactivity such as thrombosis, platelet aggregates are frequently detected in citrated blood samples using a scattergram of a hematology analyzer. This study aimed to evaluate the effects of platelet aggregate formation on light transmission aggregometry results. METHODS: We used 19 citrated blood samples in which platelet aggregate formation was intentionally induced by a hematology analysis process. Employing fully automated light transmission aggregometry and agonists including adenosine diphosphate or collagen, light transmission aggregometry maximum aggregation percentage, platelet count, and mean platelet volume of platelet-rich plasma before and after platelet aggregate formation were evaluated. RESULTS: Light transmission aggregometry maximum aggregation percentage with adenosine diphosphate or collagen was significantly lower in the samples after than before platelet aggregate formation. Platelet count and mean platelet volume were both decreased by platelet aggregate formation (P < .01), suggesting that maximum aggregation percentage reduction was caused by the decrease in activated large platelets in the platelet-rich plasma. CONCLUSION: This study clarified that platelet aggregate formation in blood samples interfered with an accurate assessment of platelet hyperactivity. To ensure reliability of light transmission aggregometry results, we must confirm that platelet aggregates have not formed in the sample, especially in those of patients with platelet hyperactivity.

Characteristic deterioration of ADAS-Jcog subscale scores and correlations with regional cerebral blood flow reductions in Alzheimer's disease.

The Alzheimer Disease Assessment Scale (Japanese version) cognitive subscale (ADAS-Jcog) is composed of a number of subscale tasks. However, it is not clear which subscale tasks are most susceptible to impairment in Alzheimer's disease (AD) or what is the relationship between reduction in regional cerebral blood flow (rCBF) and decreased ADAS-Jcog scores. Subjects were 32 AD patients, aged 52-86 years. We examined the relationship between subscale tasks that showed marked score changes and brain regions that showed reduced rCBF over a 2-year period. rCBF was measured by single-photon emission computed tomography (SPECT) with technetium-99m ethyl cysteinate dimer (99mTc-ECD), and the SPECT imaging data were analyzed with the easy Z-score imaging system (eZIS) and voxel-based stereotactic extraction estimation (vbSEE) methods. Total score of ADAS-Jcog deteriorated from 19.5 ± 7.0 to 35.7 ± 15.2 after 2 years. Subscale scores were significantly worse in all fields, particularly in orientation, word recall, remembering test instructions, commands, constructional praxis, and ideational praxis, in that order. Significant correlations were found between (1) word recall and commands and rCBF in the left middle temporal lobe, (2) naming objects/fingers and rCBF in the left temporal (middle, inferior) lobe, and (3) constructional and ideational praxis and rCBF in the right parietal (superior, inferior) lobe, temporal (superior, middle) lobe, angular gyrus, and cingulate gyrus. We identified the brain regions associated with specifically impaired subscales of ADAS-Jcog during progressive deterioration of AD over 2 years.

Low Transthyretin Levels Predict Poor Prognosis in Cancer Patients in Palliative Care Settings.

OBJECTIVES: Although transthyretin (TTR) is a nutritional indicator and is influenced by systemic inflammation, it may be a good prognostic indicator for cancer patients in palliative care settings. This study investigates the correlation between low TTR levels and survival among cancer patients in palliative care settings. METHODS: This was a sub-analysis of a prospective, multicenter cohort study. Patients who had advanced-stage cancer and who were newly referred to palliative care services were eligible to participate; however, those receiving anti-tumor therapy were excluded. Survival analyses were performed to clarify predictors of poor prognosis. RESULTS: A total of 144 patients were enrolled (45.1% female; median age, 72 years). Cox regression analysis revealed that low TTR levels (<10.9 mg/l) (hazard ratio 1.74, P = 0.025), poor muscle power (1.71, P = 0.045), and fatigue (1.89, P = 0.024) were predictors of poor prognosis. Median survival in patients with low TTR levels (<10.9 mg/l) was 26 days, which was significantly shorter than those with high TTR levels (≥10.9 mg/l) (50 days; P < 0.001). CONCLUSION: Low TTR levels may be indicators for poor prognosis among cancer patients in palliative care settings.

Dual Therapy with Aspirin and Cilostazol May Improve Platelet Aggregation in Noncardioembolic Stroke Patients: A Pilot Study.

Objective Some previous studies have found clinical benefit of dual antiplatelet therapy with aspirin and cilostazol for prevention of secondary stroke, but the physiological mechanism involved remains unknown. We aimed to clarify the effects of aspirin/cilostazol therapy on the platelet and endothelial functions of patients with acute noncardioembolic ischemic stroke, in comparison to patients who were treated with aspirin alone. Methods The present randomized prospective pilot study enrolled 24 patients within a week after the onset of noncardioembolic ischemic stroke. The patients were randomly allocated to receive aspirin (100 mg/day) (A group; 11 patients) or cilostazol (200 mg/day) plus aspirin (100 mg/day) (CA group; 13 patients). We measured platelet aggregation, platelet activation, and the thrombomodulin (TM), highly sensitive C-reactive protein (hs-CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand (vWF) antigen levels and vWF activity over a 4-week period after enrollment. Results There was no significant difference in the platelet functions of the A and CA groups. However, the platelet aggregation induced by adenosine diphosphate (ADP) was decreased at 2 and 4 weeks (p<0.05) after treatment in comparison to the pre-treatment values in the CA group, but not in the A group. Platelet activation, and the hs-CRP, TM, ICAM-1, VCAM-1 and vWF values did not significantly decrease after treatment in either group. Conclusion Although there were no significant differences in platelet aggregation, platelet activation or the endothelial biomarker levels of the A and CA groups, dual therapy with aspirin and cilostazol inhibited platelet aggregation in comparison to the pre-treatment values, similarly to patients who received aspirin alone. This may suggest the clinical usefulness of dual therapy with aspirin and cilostazol in the treatment of patients with noncardioembolic ischemic stroke.

Complement component 5 promotes lethal thrombosis.

Extracellular histones promote platelet aggregation and thrombosis; this is followed by induction of coagulation disorder, which results in exhaustion of coagulation factors. Complement component 5 (C5) is known to be associated with platelet aggregation and coagulation system activation. To date, the pathological mechanism underlying liver injury has remained unclear. Here, we investigated whether C5 promotes liver injury associated with histone-induced lethal thrombosis. C5-sufficient and C5-deficient mice received single tail vein injections of purified, unfractionated histones obtained from calf thymus (45-75 µg/g). Subsequently, the mice were monitored for survival for up to 72 h. Based on the survival data, the 45 µg/g dose was used for analysis of blood cell count, liver function, blood coagulation ability, and promotion of platelet aggregation and platelet/leukocyte aggregate (PLA) production by extracellular histones. C5-deficient mice were protected from lethal thrombosis and had milder thrombocytopenia, consumptive coagulopathy, and liver injury with embolism and lower PLA production than C5-sufficient mice. These results indicate that C5 is associated with coagulation disorders, PLA production, and embolism-induced liver injury. In conclusion, C5 promotes liver injury associated with histone-induced lethal thrombosis.

Cilostazol protects against microvascular brain injury in a rat model of type 2 diabetes.

Cilostazol, a pluripotent phosphodiesterase III-specific inhibitor with anti-platelet and vasculogenic effects, is useful for preventing recurrent brain vascular events, particularly in stroke patients with diabetes mellitus (DM). However, it is unclear whether cilostazol affects autoregulatory responses in small cerebral arteries. Thus, we investigated the effect of cilostazol on diabetic brain vasculopathy in a model of type II DM using male OLETF rats. OLETF rats were treated with either cilostazol (CG) or vehicle (VG) and subjected to microangiography with monochromatic synchrotron radiation to investigate middle cerebral artery (MCA) vasoreactivity following an injection of acetylcholine (Ach). Ach administration led to MCA diameter contraction in the VG, but MCA dilation in the CG. We also evaluated morphological changes in the small intracranial vessels and found that in the CG, the endothelial cell structure in the small artery was not destroyed. Moreover, protein levels of phosphorylated endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were higher in each evaluated brain region in CG rats vs. VG rats. Our results suggest that cilostazol could potentially improve autoregulatory responses in the small cerebral arteries by increasing eNOS phosphorylation and VEGF expression in DM, and thus, may act as a neurovascular protectant.

Clinical Utility of Platelet Function Testing Following Non-Cardioembolic Stroke.

OBJECTIVE: To verify the usefulness in selection of antiplatelet agent based on the platelet functional assays on the secondary prevention of ischemic stroke. METHODS: Platelet functional assays were performed twice for acute ischemic stroke patients at hospitalization and 1 month after. An antiplatelet agent was administered based on the results of initial assay. The alterations of platelet aggregation by antiplatelet agent were evaluated in the second assay, and the patients were subsequently divided into inhibited and invariance groups. The relationship between incidence of recurrent ischemic or hemorrhagic stroke and the alterations of platelet aggregation by each selected antiplatelet agent was assessed. RESULTS: Of the 585 consecutive patients, 124 were enrolled in the present study. Recurrent ischemic stroke was seen in 6 (5.3%) and 2 (18.2%) patients in the inhibited and invariance groups during the study period, respectively. In patients who were observed for more than 12 months, recurrent ischemic stroke was seen in 4 (5.0%) and 2 (33.3%) patients in the inhibited and invariance groups, respectively (p = 0.009). CONCLUSIONS: We indicated that selection of the optimum antiplatelet agent based on the platelet functional assays for each individual patient may contribute to a reduction in the incidence of recurrence of ischemic stroke.

Surprise Questions for Survival Prediction in Patients With Advanced Cancer: A Multicenter Prospective Cohort Study.

BACKGROUND: Predicting the short-term survival in cancer patients is an important issue for patients, family, and oncologists. Although the prognostic accuracy of the surprise question has value in 1-year mortality for cancer patients, the prognostic value for short-term survival has not been formally assessed. The primary aim of the present study was to assess the prognostic value of the surprise question for 7-day and 30-day survival in patients with advanced cancer. PATIENTS AND METHODS: The present multicenter prospective cohort study was conducted in Japan from September 2012 through April 2014, involving 16 palliative care units, 19 hospital-based palliative care teams, and 23 home-based palliative care services. RESULTS: We recruited 2,425 patients and included 2,361 for analysis: 912 from hospital-based palliative care teams, 895 from hospital palliative care units, and 554 from home-based palliative care services. The sensitivity, specificity, positive predictive value, and negative predictive value of the 7-day survival surprise question were 84.7% (95% confidence interval [CI], 80.7%-88.0%), 68.0% (95% CI, 67.3%-68.5%), 30.3% (95% CI, 28.9%-31.5%), and 96.4% (95% CI, 95.5%-97.2%), respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for the 30-day surprise question were 95.6% (95% CI, 94.4%-96.6%), 37.0% (95% CI, 35.9%-37.9%), 57.6% (95% CI, 56.8%-58.2%), and 90.4% (95% CI, 87.7%-92.6%), respectively. CONCLUSION: Surprise questions are useful for screening patients for short survival. However, the high false-positive rates do not allow clinicians to provide definitive prognosis prediction. IMPLICATIONS FOR PRACTICE: The findings of this study indicate that clinicians can screen patients for 7- or 30-day survival using surprise questions with 90% or more sensitivity. Clinicians cannot provide accurate prognosis estimation, and all patients will not always die within the defined periods. The screened patients can be regarded as the subjects to be prepared for approaching death, and proactive discussion would be useful for such patients.

The Accuracy of Physicians' Clinical Predictions of Survival in Patients With Advanced Cancer.

CONTEXT: Accurate prognoses are needed for patients with advanced cancer. OBJECTIVES: To evaluate the accuracy of physicians' clinical predictions of survival (CPS) and assess the relationship between CPS and actual survival (AS) in patients with advanced cancer in palliative care units, hospital palliative care teams, and home palliative care services, as well as those receiving chemotherapy. METHODS: This was a multicenter prospective cohort study conducted in 58 palliative care service centers in Japan. The palliative care physicians evaluated patients on the first day of admission and followed up all patients to their death or six months after enrollment. We evaluated the accuracy of CPS and assessed the relationship between CPS and AS in the four groups. RESULTS: We obtained a total of 2036 patients: 470, 764, 404, and 398 in hospital palliative care teams, palliative care units, home palliative care services, and chemotherapy, respectively. The proportion of accurate CPS (0.67-1.33 times AS) was 35% (95% CI 33-37%) in the total sample and ranged from 32% to 39% in each setting. While the proportion of patients living longer than CPS (pessimistic CPS) was 20% (95% CI 18-22%) in the total sample, ranging from 15% to 23% in each setting, the proportion of patients living shorter than CPS (optimistic CPS) was 45% (95% CI 43-47%) in the total sample, ranging from 43% to 49% in each setting. CONCLUSION: Physicians tend to overestimate when predicting survival in all palliative care patients, including those receiving chemotherapy.

Effect of single nucleotide polymorphisms of the prostacyclin receptor gene on platelet activation in Japanese healthy subjects and patients with cerebral infarction.

Cerebral infarction (CI) is a complex multifactorial disorder that is thought to result from the interaction of various environmental factors and an individual's genetic make-up, including genes associated with platelet activation. In order to clarify whether single nucleotide polymorphisms (SNPs) of the prostacyclin receptor (IP) gene affects platelet activation in ischemic stroke, we investigated the relationship between platelet function and genetic polymorphism of the coding sequence of the IP gene in 64 Japanese patients with CI and 54 healthy subjects. We determined the entire nucleotide sequence of the IP gene in healthy Japanese subjects, and found that an adenine (A) to cytosine (C) substitution at base 984 (A984C) in exon 3 is the most frequent SNP. Using flow cytometry, the power-transformed mean percentage of PAC-1-positive platelets, [PAC-1](1/3), was significantly higher in healthy subjects with the C/C genotype than in healthy subjects with the A/A genotype (p ≤ 0.05), although there was no significant difference in patients with CI between these two genotypes. Furthermore, we genotyped 158 control patients and 106 patients with CI. The homozygous C/C genotype was more frequently found in the CI group (46.2%) than in the healthy control group (17.1%; p < 0.001). The present report is the first to show an association between the A984C polymorphism of the IP gene and platelet activation in Japanese subjects. This polymorphism may be clinically significant in disorders in which prostacyclin plays a key role, such as CI.

Appearance of WBC-platelet complex in acute ischemic stroke, predominantly in atherothrombotic infarction.

AIM: Platelet aggregates with white blood cells (WBC-platelet complex) have recently been proposed as a marker of activated platelets, in addition to well-known molecular markers. We aimed to investigate the colocalization of activated platelets and WBC-platelet complex by means of flow cytometry, in patients with ischemic stroke. METHODS: Eighty-six patients with cerebral infarction (CI) in the acute phase (58 males, 28 females; 65±14 years old) and 62 non-CI controls (23 males, 39 females; 53±14 years old) were registered. The appearance of WBC-platelet complex was quantified using 3-color flow cytometry. RESULTS: The appearance rate of WBC-platelet complex was significantly higher in the CI group than in the controls. The appearance rate of WBC-platelet complex was significantly higher in atherothrombotic infarction (AT) than in lacunar infarction (LA) (p < 0.05). Furthermore, positive rates of both monocyte-platelet complex and granulocyte-platelet complex, but not lymphocyte-platelet complex, were significantly higher in the AT group than in the controls. CONCLUSION: We concluded that WBC-platelet complex, especially involving monocytes and granulocytes, is a novel marker of platelet activation in the acute phase of ischemic stroke, mainly in AT.

Significant relationship between platelet activation and apnea-hypopnea index in patients with obstructive sleep apnea syndrome.

Obstructive sleep apnea syndrome (OSAS) is an independent risk factor for arterial thrombosis, which is associated with high cardiovascular morbidity and mortality. To investigate the possible involvement of activated platelets, we evaluated the relationship between severity of OSAS and appearance of platelet aggregates (a marker of activated platelets) in 35 OSAS patients. Platelet aggregates were quantitatively determined by means of flow cytometry. There was a significant correlation between platelet aggregates and apnea-hypopnea index in the severe (AHI≥30 events/hour) group (r=0.756, p<0.001), but not in the mild-moderate (5≤AHI<30 events/hour) group (r=-0.032, p=0.905). The results indicate that the appearance of platelet aggregates increases with an increase in the severity of OSAS.