Reports of thrombosis post coronavirus disease 2019 (COVID-19) mRNA vaccination have sparked concerns about the safety of these immunizations. As of October 31, 2021, the Health Sciences Authority of Singapore reported 13 suspected cases of cerebral venous thrombosis (CVT) with the Pfizer-BioNTech/Comirnaty (BNT162b2) and Moderna/Spikevax (mRNA-1273) COVID-19 vaccines out of 9 953 673 total number of doses administered.¹

We previously reported on three patients who developed CVT post BNT162b2 vaccination, occurring 1–9 days after the second dose.² Compared to vaccine-induced thrombotic thrombocytopenia (VITT) associated with the use of adenovirus vector ChAdOx1 nCoV-19 and Ad26.COV2.S COVID-19 vaccines, these patients with CVT post BNT162b2 vaccine were negative for antiplatelet factor 4 (PF4) antibodies. In a study of 30 healthcare workers who received the BNT162b2 vaccine, no hypercoagulable state was found.³ Their coagulation parameters remained unchanged postvaccination except for a slight increase in platelet levels 14 days after the second dose of BNT162b2 vaccine. Similarly, no differences were detected in thromboelastometry, thrombin generation, thrombin receptor activating peptide, adenosine diphosphate, and arachidonic acid-induced platelet aggregation tests after first dose of the BNT162b2 or the ChAdOx1 vaccines in healthy volunteers by Campello et al.⁴

No study to date has evaluated markers for endothelial activation post mRNA vaccination. Virchow's triad of venous stasis, hypercoagulable state, and endothelial dysfunction summarizes the pathophysiologic mechanisms leading to thrombosis. SARS-CoV-2 infection is characterized by COVID-19 associated coagulopathy, evidenced by elevated D-dimer, Von Willebrand factor (vWF), Factor VIII levels, hyperfibrinogenemia in critically ill patients.^{5, 6} Endothelial cell adhesion molecules, including serum levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (V-CAM1) were elevated in severe COVID-19 infection.⁷ We hypothesized that post BNT162b2 vaccination, markers of endothelial activation as well as parameters of coagulation may be elevated. A prospective, observational, pilot study was performed to evaluate the endothelial and coagulation profile in a series of healthy participants who had received two doses of the BNT162b2 mRNA vaccine, with the aim to determine if the BNT162b2 vaccination results in endothelial activation or hypercoagulability by studying the endothelial adhesion molecules and coagulation parameters pre and post mRNA vaccination.

Eighteen participants who received the BNT162b2 vaccine were enrolled in this study. Participants completed a questionnaire on their cardiovascular and thrombotic risk factors, including the chronic medications they were taking prior to vaccination. All participants had three blood samples planned: prevaccination, after first dose of BNT162b2 vaccine, and after second dose of BNT162b2 vaccine. The median age of the participants was 35 years (interquartile range [IQR 31–44]), and 14 (78%) were female. Fifteen participants did not have any cardiovascular or thrombotic risk factors. Two reported a medical history of hypertension and one had a history of stroke; and they were on antihypertensive medications and antiplatelet therapy, respectively, during the period of vaccination and blood taking. All 18 participants completed two doses of BNT162b2 vaccination, with the second dose of BNT162b2 administered a median of 21 (IQR 21–22) days after the first dose. All tolerated the vaccination with no serious adverse reaction and no thrombotic events. Blood samples were collected at three-time points: prevaccination (on day of vaccination), a median of 17 (IQR 16–18) days after the first dose of BNT162b2 vaccine, and a median of 9 (IQR 7.5–14.5) days after the second dose of BNT162b2 vaccine. Only one participant defaulted the blood sampling after the first BNT162b2 vaccine dose but completed prespecified blood sampling prevaccination and post-second dose of vaccine.

The following biomarkers were assayed by enzyme-linked immunosorbent assay: ICAM-1, VCAM-1, and P-selectin (all R&D Systems, Abrington, UK). Coagulation tests were performed using the STA R Max Series coagulation analyzer (Diagnostica Stago, France) and Sysmex CN-6000 automated coagulation analyzer (Sysmex Corporation, Kobe, Japan). Prothrombin time (PT) was measured with Innovin (Siemens Healthcare, Marburg, Germany), activated partial thromboplastin time (aPTT) with Dade Actin FSL (Siemens Healthcare), fibrinogen (modified Clauss) with STA Liquid FIB, D-dimer with STA Liatest D-Dimer. Clotting factor levels (Factor VIII) were measured with STA Deficient VIII. vWF antigen was assayed with an immunoturbidimetric method using STA Liastest vWF: Ag kit. Clot waveform analysis (CWA) was performed with Sysmex CN-6000 automated coagulation analyzer (Sysmex Corporation) with parameters obtained from aPTT and for PT, as per International Society of Hemostasis and Thrombosis Scientific and Standardization Committee recommendation. Four quantitative parameters were recorded—“Min1” (maximum velocity), “Min2” (maximum acceleration), “Max2” (maximum deceleration), and “Delta change” (difference between initial maximum and final maximum values of light transmittance). For continuous data, median and IQR were presented due to its skewed distribution. Tests of association between coagulation and endothelial parameters were performed. Normality of the data was assessed using histogram and Shapiro–Wilk's test. The data were transformed if it was shown to be skewed. Linear mixed models were used to estimate the difference in blood markers between two-time points, treating participants as a random effect. As the study population comprised only 18 participants, no adjustment was made in the model. The repeated measures analysis of variance was also used to analyze the same data, as part of sensitivity analyses. The Mauchly's test of sphericity was assessed as part of the analysis; if the p-value based on the Mauchly's test was <.05, the p-value based on Greenhouse–Geisser would be reported in the test of differences of blood markers over time. Analyses were performed using STATA 17 (StataCorp 2021. Stata Statistical Software: Release 17, College Station, TX: StataCorp LLC.). Statistical significance was declared if a 2-sided p-value < .05. Bonferroni correction was used in instances of multiple comparisons.

Our results show no evidence of endothelial activation (ICAM, VCAM-1, and P-selectin) or hypercoagulability, with the median values of endothelial cell adhesion molecules and coagulation parameters remaining within normal limits pre and postvaccination (Table 1). There was a statistically significant increase in median ICAM levels post first and second dose of vaccination, although this remained below the normal limit of ICAM levels. A statistically significant decrease in PT and aPTT was observed postvaccination, with a corresponding increase in aPTT CWA for maximum acceleration (max2) and maximum deceleration (max2) when comparing the differences in median values post first and second dose of vaccination with prevaccination levels. However, these parameters remain within the reference ranges for PT, aPTT, and CWA. Even though we did not capture data on the reactogenicity of mRNA vaccination in our participants, which encompasses manifestations of the inflammatory response to vaccination such as injection-site pain, redness, or induration, as well as systemic symptoms such as fever, we postulate that these mild variations in endothelial markers and coagulation parameters, though statistically significant, may be related to a local inflammatory immune response to vaccination. While patients with moderate to severe COVID-19 have increased hypercoagulability and endothelial activation, with an increased incidence of thrombosis, the localized expression of the spike protein with mRNA vaccination does not result in severe inflammation that can cause a hypercoagulable state in healthy subjects, as demonstrated by our study data from our study and those of the two previous studies mentioned,^{3, 4} or excessive endothelial activation as shown by our study. One participant who did not report any cardiovascular risk factors demonstrated raised vWF Ag and Factor VIII levels of 181% and 198%, respectively, prior to vaccination. Her levels of vWF Ag dropped to 163% 16 days after the first dose of BNT162b2 vaccine and 179% 9 days after the second dose of BNT162b2 vaccine while her levels of Factor VIII were 184% and 183% after the first and second dose of BNT162b2 vaccine, respectively. Another participant with a history of stroke did not demonstrate significant baseline levels or increase in her coagulation parameters nor upregulation of endothelial adhesion molecules.

There are several limitations of our study. First, the small sample size may not be representative of the larger population. Second, our study focused on only the BNT162b2 vaccine and hence the results are not generalizable to all COVID-19 vaccines as VITT is reported to be more strongly associated with adenovirus vector vaccines than mRNA vaccines.⁸ Third, majority of our study participants were younger, healthy individuals without any cardiovascular risk factors, and it is unclear if the results will be similar in a population with such risk factors. Lastly, we did not evaluate markers of inflammation such as C-reactive protein, interleukin-6, or tumor necrosis factor, which would be important in establishing any postvaccination increase in local or systemic inflammation, or tests of platelet function to detect postvaccination platelet activation.

In conclusion, our findings provide reassuring preliminary data that BNT162b2 vaccination does not result in endothelial activation or a hypercoagulable state. While the definition and mechanism of VITT are more clearly defined and linked to the presence of PF4 antibodies, the cause of rare non-VITT thrombosis remains elusive.^{9, 10} Additional studies will be required to identify the population at risk of vaccine-associated thrombosis and how to monitor for this rare but serious complication.

有关 2019 年冠状病毒病 (COVID-19) mRNA 疫苗接种后血栓形成的报告引发了对这些免疫接种安全性的担忧。截至 2021 年 10 月 31 日，新加坡卫生科学局报告了 13 例疑似脑静脉血栓形成 (CVT) 病例，使用 Pfizer-BioNTech/Comirnaty (BNT162b2) 和 Moderna/Spikevax (mRNA-1273) COVID-19 疫苗（共 9 种）给药剂量总数为 953 673 次。¹

我们之前报道了三名在 BNT162b2 疫苗接种后发生 CVT 的患者，发生在第二剂疫苗后 1-9 天。²与使用腺病毒载体 ChAdOx1 nCoV-19 和 Ad26.COV2.S COVID-19 疫苗相关的疫苗引起的血栓性血小板减少症 (VITT) 相比，这些接受 BNT162b2 疫苗后 CVT 的患者抗血小板因子 4 (PF4) 抗体呈阴性. 在对接受 BNT162b2 疫苗的 30 名医护人员进行的一项研究中，未发现高凝状态。³除了在第二剂 BNT162b2 疫苗后 14 天血小板水平略有增加外，他们的凝血参数在接种后保持不变。同样，Campello 等人在健康志愿者中首次接种 BNT162b2 或 ChAdOx1 疫苗后，在血栓弹力图、凝血酶生成、凝血酶受体激活肽、二磷酸腺苷和花生四烯酸诱导的血小板聚集试验中未检测到差异。⁴

迄今为止，还没有研究评估 mRNA 疫苗接种后内皮激活的标志物。Virchow 的静脉淤滞、高凝状态和内皮功能障碍三联征总结了导致血栓形成的病理生理机制。SARS-CoV-2 感染的特征是与 COVID-19 相关的凝血病，表现为危重患者的 D-二聚体、血管性血友病因子 (vWF)、因子 VIII 水平、高纤维蛋白原血症升高。^{5, 6}内皮细胞粘附分子，包括细胞间粘附分子-1 (ICAM-1) 和血管细胞粘附分子-1 (V-CAM1) 的血清水平在严重的 COVID-19 感染中升高。⁷我们假设 BNT162b2 疫苗接种后，内皮激活标志物以及凝血参数可能会升高。进行了一项前瞻性、观察性、初步研究，以评估一系列接受两剂 BNT162b2 mRNA 疫苗的健康参与者的内皮和凝血特征，旨在通过研究确定 BNT162b2 疫苗接种是否导致内皮激活或高凝状态mRNA疫苗接种前后的内皮粘附分子和凝血参数。

接受 BNT162b2 疫苗的 18 名参与者参加了这项研究。参与者完成了一份关于他们的心血管和血栓形成风险因素的问卷，包括他们在接种疫苗前服用的慢性药物。所有参与者都计划了三个血液样本：疫苗接种前、第一剂 BNT162b2 疫苗后和第二剂 BNT162b2 疫苗后。参与者的中位年龄为 35 岁（四分位距 [IQR 31-44]），其中 14 人（78%）为女性。15 名参与者没有任何心血管或血栓形成危险因素。两人报告有高血压病史，一人有中风病史；在疫苗接种和采血期间，他们分别接受了抗高血压药物和抗血小板治疗。所有 18 名参与者都完成了两剂 BNT162b2 疫苗接种，第二剂 BNT162b2 在第一剂后的中位数为 21 (IQR 21-22) 天。所有人都能耐受疫苗接种，没有严重的不良反应，也没有血栓形成事件。在三个时间点采集血样：接种前（接种当天）、第一剂 BNT162b2 疫苗后 17（IQR 16-18）天的中位数和接种后 9（IQR 7.5-14.5）天的中位数第二剂 BNT162b2 疫苗。只有一名参与者在第一剂 BNT162b2 疫苗接种后未进行血液采样，但完成了预先指定的血液采样预接种和第二剂疫苗后。所有人都能耐受疫苗接种，没有严重的不良反应，也没有血栓形成事件。在三个时间点采集血样：接种前（接种当天）、第一剂 BNT162b2 疫苗后 17（IQR 16-18）天的中位数和接种后 9（IQR 7.5-14.5）天的中位数第二剂 BNT162b2 疫苗。只有一名参与者在第一剂 BNT162b2 疫苗接种后未进行血液采样，但完成了预先指定的血液采样预接种和第二剂疫苗后。所有人都能耐受疫苗接种，没有严重的不良反应，也没有血栓形成事件。在三个时间点采集血样：接种前（接种当天）、第一剂 BNT162b2 疫苗后 17（IQR 16-18）天的中位数和接种后 9（IQR 7.5-14.5）天的中位数第二剂 BNT162b2 疫苗。只有一名参与者在第一剂 BNT162b2 疫苗接种后未进行血液采样，但完成了预先指定的血液采样预接种和第二剂疫苗后。

通过酶联免疫吸附测定法测定以下生物标志物：ICAM-1、VCAM-1, 和 P-选择素 (所有 R&D Systems, Abrington, UK)。使用 STA R Max 系列凝血分析仪（Diagnostica Stago，法国）和 Sysmex CN-6000 自动凝血分析仪（Sysmex Corporation，Kobe，Japan）进行凝血测试。凝血酶原时间 (PT) 用 Innovin (Siemens Healthcare, Marburg, Germany) 测量，活化部分凝血活酶时间 (aPTT) 用 Dade Actin FSL (Siemens Healthcare)，纤维蛋白原 (改良 Clauss) 用 STA Liquid FIB，D-二聚体用 STA Liatest D-二聚体。用 STA Deficient VIII 测量凝血因子水平（因子 VIII）。使用 STA Liastest vWF: Ag 试剂盒通过免疫比浊法测定 vWF 抗原。使用 Sysmex CN-6000 自动凝血分析仪（Sysmex Corporation）进行凝块波形分析（CWA），参数从 aPTT 和 PT 获得，根据国际止血和血栓形成科学与标准化委员会的建议。记录了四个定量参数——“Min1”（最大速度）、“Min2”（最大加速度）、“Max2”（最大减速度）和“Delta 变化”（透光率的初始最大值和最终最大值之间的差异）。对于连续数据，中位数和 IQR 由于其偏态分布而呈现。进行了凝血和内皮参数之间关联的测试。使用直方图和夏皮罗-威尔克检验评估数据的正态性。如果数据显示有偏差，则数据将被转换。线性混合模型用于估计两个时间点之间血液标志物的差异，将参与者视为随机效应。由于研究人群仅包括 18 名参与者，模型中没有进行任何调整。作为敏感性分析的一部分，重复测量方差分析也用于分析相同的数据。Mauchly 的球形度测试作为分析的一部分进行了评估；如果基于 Mauchly 检验的 p 值 <.05，基于Greenhouse-Geisser 的 p 值将在血液标志物随时间差异的检验中报告。使用 STATA 17 (StataCorp 2021.Stata Statistical Software: Release 17, College Station, TX: StataCorp LLC.) 进行分析。如果 2 边p值 < .05 ，则声明统计显着性。在多重比较的情况下使用 Bonferroni 校正。

我们的结果显示没有证据表明内皮细胞活化（ICAM、VCAM-1 和 P-选择素）或高凝状态，内皮细胞粘附分子和凝血参数的中值在疫苗接种前后保持在正常范围内（表 1）。在第一剂和第二剂疫苗接种后，中位 ICAM 水平在统计学上显着增加，尽管这仍然低于 ICAM 水平的正常限度。接种疫苗后观察到 PT 和 aPTT 有统计学意义的下降，当将第一剂和第二剂疫苗接种后的中值差异与疫苗接种前水平进行比较时，最大加速度 (max2) 和最大减速度 (max2) 的 aPTT CWA 相应增加。但是，这些参数仍然在 PT、aPTT 和 CWA 的参考范围内。尽管我们没有在我们的参与者中获得有关 mRNA 疫苗接种反应原性的数据，其中包括对疫苗接种的炎症反应的表现，例如注射部位疼痛、发红或硬结，以及发烧等全身症状，但我们假设内皮标志物和凝血参数的这些轻微变化虽然具有统计学意义，但可能与对疫苗接种的局部炎症免疫反应有关。虽然中度至重度 COVID-19 患者的高凝状态和内皮激活增加，血栓形成的发生率增加，但 mRNA 疫苗接种后刺突蛋白的局部表达不会导致严重的炎症，从而导致健康受试者出现高凝状态，如我们的研究所示， ^{3, 4或过度的内皮激活。}一名未报告任何心血管危险因素的参与者在接种疫苗前证明 vWF Ag 和因子 VIII 水平分别提高了 181% 和 198%。在第一剂 BNT162b2 疫苗后 16 天，她的 vWF Ag 水平下降至 163%，在第二剂 BNT162b2 疫苗后 9 天降至 179%，而在第一剂和第二剂 BNT162b2 后，她的因子 VIII 水平分别为 184% 和 183%疫苗，分别。另一名有中风病史的参与者没有表现出显着的基线水平或凝血参数的增加，也没有表现出内皮粘附分子的上调。

我们的研究有几个局限性。首先，小样本量可能无法代表较大的人群。其次，我们的研究仅关注 BNT162b2 疫苗，因此结果不能推广到所有 COVID-19 疫苗，因为据报道 VITT 与腺病毒载体疫苗的相关性比 mRNA 疫苗更强。⁸第三，我们的大多数研究参与者都是年轻、健康的个体，没有任何心血管风险因素，目前尚不清楚在有这些风险因素的人群中结果是否相似。最后，我们没有评估炎症标志物，如 C 反应蛋白、白细胞介素 6 或肿瘤坏死因子，这对于确定疫苗接种后局部或全身炎症的任何增加或检测血小板功能以检测疫苗接种后血小板活化很重要.

总之，我们的研究结果提供了令人放心的初步数据，即 BNT162b2 疫苗接种不会导致内皮激活或高凝状态。虽然 VITT 的定义和机制更加明确，并且与 PF4 抗体的存在有关，但罕见的非 VITT 血栓形成的原因仍然难以捉摸。^{9, 10}需要进一步的研究来确定有疫苗相关血栓形成风险的人群以及如何监测这种罕见但严重的并发症。