Fibrin drives thromboinflammation and neuropathology in COVID-19.

Life-threatening thrombotic events and neurological symptoms are prevalent in COVID-19 and are persistent in patients with long COVID experiencing post-acute sequelae of SARS-CoV-2 infection1-4. Despite the clinical evidence1,5-7, the underlying mechanisms of coagulopathy in COVID-19 and its consequences in inflammation and neuropathology remain poorly understood and treatment options are insufficient. Fibrinogen, the central structural component of blood clots, is abundantly deposited in the lungs and brains of patients with COVID-19, correlates with disease severity and is a predictive biomarker for post-COVID-19 cognitive deficits1,5,8-10. Here we show that fibrin binds to the SARS-CoV-2 spike protein, forming proinflammatory blood clots that drive systemic thromboinflammation and neuropathology in COVID-19. Fibrin, acting through its inflammatory domain, is required for oxidative stress and macrophage activation in the lungs, whereas it suppresses natural killer cells, after SARS-CoV-2 infection. Fibrin promotes neuroinflammation and neuronal loss after infection, as well as innate immune activation in the brain and lungs independently of active infection. A monoclonal antibody targeting the inflammatory fibrin domain provides protection from microglial activation and neuronal injury, as well as from thromboinflammation in the lung after infection. Thus, fibrin drives inflammation and neuropathology in SARS-CoV-2 infection, and fibrin-targeting immunotherapy may represent a therapeutic intervention for patients with acute COVID-19 and long COVID.

Modification with CREKA Improves Cell Retention in a Rat Model of Myocardial Ischemia Reperfusion.

Poor cell homing limits the efficacy of cardiac cellular therapy. The homing peptide, cysteine-arginine-glutamic acid-lysine-alanine (CREKA), targets fibrin effectively which is involved in the repair process of tissue injury. Here, we assessed if CREKA-modified stem cells had enhanced fibrin-mediated homing ability resulting in better functional recovery and structural preservation in a rat myocardial injury model. CREKA-modified mesenchymal stem cells (CREKA-MSCs) were obtained via membrane fusion with CREKA-modified liposomes. The fibrin targeting ability of CREKA-MSCs was examined both in vitro and in vivo. Under both static and flow conditions in vitro, CREKA significantly enhanced MSCs binding ability to fibrin clots (2.6- and 2.3-fold, respectively). CREKA-MSCs showed 6.5-fold higher accumulation than unmodified MSCs in injured rat myocardium one day after administration, resulting in better structural preservation and functional recovery. Fibrin is, therefore, a novel target for enhancing homing of transplanted cells to injured myocardium, and the delivery system of fibrin-targeting is on behalf of a universalizable platform technology for regenerative medicine. Stem Cells 2019;37:663-676.

Favorable 2'-substitution in the loop region of a thrombin-binding DNA aptamer.

Simple 2'-OMe-chemical modification in the loop region of the 15mer G-rich DNA sequence GGTTGGTGTGGTTGG is reported. The G-quadruplex structure of this thrombin-binding aptamer (TBA), is stabilized by single modifications (T → 2'-OMe-U), depending on the position of the modification. The structural stability also renders significantly increased inhibition of thrombin-induced fibrin polymerization, a process closely associated with blood-clotting.

Comparative Effect of Quercetin and Quercetin-3-O-ß-d-Glucoside on Fibrin Polymers, Blood Clots, and in Rodent Models.

The present study evaluates the in vitro, in vivo, and ex vivo antithrombotic and anticoagulant effect of two flavonoids: quercetin and quercetin-3-O-ß-d-glucoside (isoquercetin). The present results have shown that quercetin and isoquercetin inhibit the enzymatic activity of thrombin and FXa and suppress fibrin clot formation and blood clotting. The prolongation effect of quercetin and isoquercetin against epinephrine and collagen-induced platelet activation may have been caused by intervention in intracellular signaling pathways including coagulation cascade and aggregation response on platelets and blood. The in vivo and ex vivo anticoagulant efficacy of quercetin and isoquercetin was evaluated in thrombin-induced acute thromboembolism model and in ICR mice. Our findings showed that in vitro and in vivo inhibitory effects of quercetin were slightly higher than that of quercetin glucoside, whereas in vitro and ex vivo anticoagulant effects of quercetin were weaker than that of quercetin glucoside because of their structural characteristics.

Spirulan from blue-green algae inhibits fibrin and blood clots: its potent antithrombotic effects.

We investigated in vitro and in vivo fibrinolytic and antithrombotic activity of spirulan and analyzed its partial biochemical properties. Spirulan, a sulfated polysaccharide from the blue-green alga Arthrospira platensis, exhibits antithrombotic potency. Spirulan showed a strong fibrin zymogram lysis band corresponding to its molecular mass. It specifically cleaved Aα and Bß, the major chains of fibrinogen. Spirulan directly decreased the activity of thrombin and factor X activated (FXa), procoagulant proteins. In vitro assays using human fibrin and mouse blood clots showed fibrinolytic and hemolytic activities of spirulan. Spirulan (2 mg/kg) showed antithrombotic effects in the ferric chloride (FeCl3 )-induced carotid arterial thrombus model and collagen and epinephrine-induced pulmonary thromboembolism mouse model. These results may be attributable to the prevention of thrombus formation and partial lysis of thrombus. Therefore, we suggest that spirulan may be a potential antithrombotic agent for thrombosis-related diseases.

A fibrinogen concentrate Haemocomplettan (Riastap) or a Factor XIII concentrate Fibrogammin combined with a mini dose of tranexamic acid can reverse the fibrin instability to fibrinolysis induced by thrombin- or FXa-inhibitor.

To assess whether Haemocomplettan(®) (fibrinogen concentrate) or Fibrogammin(®) (Factor XIII concentrate) can be used to manage bleeding complications of antithrombotic treatment, we examined a normal plasma pool spiked with AR-H067637 (thrombin inhibitor) or rivaroxaban (activated factor X-inhibitor), to which one of the concentrates was added. Fibrin network permeability (Ks), images of Scanning Electron Microscopy (SEM) and Clot Lysis Time (CLT) were examined. Both inhibitors increased the Ks levels, which could be fully or partly reversed by Haemocomplettan(®) or Fibrogammin(®) respectively. However, these modified clots with tightened network remained non-resistant to fibrinolysis, shown as unaffected CLT. Tranexamic acid at a very low concentration (0·4 mg/ml) aided the two concentrates to stabilize the clots, where the prolongation of CLT was more pronounced for a lower dose than a higher dose of Haemocomplettan(®) while Fibrogammin(®) brought the greatest delay to CLT out of all additions. These observations were partly supported by SEM images, displaying alterations of fibrin fibre arrangement known to influence fibirinolysis. The in vitro data suggest that Haemocomplettan(®) or Fibrogammin(®) given in combination with a mini dose of tranexamic acid may slow down the natural clearance of fibrin clot by plasmin and thus prevent patients from haemorrhagic complications during antithrombotic therapy.

[Dependence of the anticoagulant activity of starch and inulin on their degree of sulfonation].

We have studied a relationship between the degree of sulfonation and anticoagulant activity of starch from Solanum tuberosum (molecular weight, 25000-30000 Da; sulfonation degree, 0.4-2.5) and inulin from Helianthus tuberosus (molecular weight, 7000-8000 Da; sulfonation degree, 0.6-1.6). Starch and inulin sulfates (i) increased the time of appearance of fibrin clots in plasma in coagulometric tests and (ii) reduced (via antithrombin) the rate of thrombin-induced hydrolysis of a chromogen substrate. The antithrombin (aIIa) activity of starch sulfates reached 16.8-70.0 IU/mg and the activity against factor Xa (aXa activity) was 2.3-16.6 IU/mg. The antithrombin activity of inulin sulfates was within 5.5-11.4 IU/mg and the activity against factor Xa (aXa activity) was within 0-1.4 IU/mg. An increase in the degree of sulfonation led to a growth in the anticoagulant activity of starch sulfates. The anticoagulant activity of starch sulfates and inulin sulfate with sulfonation degree 1.0 is mediated by antithrombin, which is the plasma inhibitor of serine proteases.

New concept of cytotoxic immunoconjugate therapy targeting cancer-induced fibrin clots.

Fibrin clots in non-malignant conditions form only at the onset or during the active stage of disease and disappear within a few weeks as a result of plasmin digestion or replacement with collagen. In contrast, fibrin clot formation and subsequent replacement with collagen in cancer persist for as long as the cancer cells survive in the body. We developed an anti-fibrin chimeric antibody that reacts with fibrin only, and not fibrinogen (the precursor of fibrin), and then attached an anticancer agent (ACA) to the antibody. Thus, the immunoconjugate did not create an immune complex in the blood stream and was selectively accumulated to fibrin clots in the tumor stroma to create a scaffold, from which effective sustained release of the ACA occurred. In a mouse model, the ACA diffused throughout the tumor tissue to damage both tumor cells and vessels, resulting in potent antitumor activity in stroma-rich spontaneous tumors. This new cancer stroma-targeting therapy may result in an increased duration of drug exposure and be a highly effective new therapy, particularly for refractory, stroma-rich cancers.

Hypochlorite-induced oxidation of fibrinogen: Effects on its thermal denaturation and fibrin structure.

BACKGROUND: Human fibrinogen, which plays a key role in plasma haemostasis, is a highly vulnerable target for oxidants. Fibrinogen undergoes posttranslational modifications that can potentially disrupt protein structure and function. METHODS: For the first time, by differential scanning calorimetry, dynamic and elastic light scattering and confocal laser scanning microscopy, the consequences of HOCl/-OCl-induced oxidation of fibrinogen on its thermal denaturation, molecular size distribution and fibrin clot network have been explored. RESULTS: Within a wide range of HOCl/-OCl concentrations (50-300 µM), the molecular size distribution remained unimodal; however, the average size of the hydrated molecules decreased. HOCl/-OCl-induced oxidation of fibrinogen resulted in the diminished thermal stability of regions D and E. As evidenced by elastic light scattering and confocal laser scanning microscopy, HOCl/-OCl caused the formation of abnormal fibrin with a decreased diameter of individual fibres. CONCLUSIONS: The current results along with data from previous studies enable one to conclude that the effect of HOCl/-OCl-mediated oxidation on the thermal stability of region D is influenced directly by oxidative damage to the D region structure. Since the E region is not subjected to oxidative modification, its structural damage is likely to be mediated by the oxidation of other protein structures, in particular α-helical coiled-coils. GENERAL SIGNIFICANCE: The experimental findings acquired in the current study could help to elucidate the consequences of oxidative stress in vivo on damage to the structure of fibrinogen/fibrin under the action of different ROS species.

Cancer stromal targeting therapy to overcome the pitfall of EPR effect.

Many animal experiments performed worldwide have proven EPR effects However, it is hard to say that the EPR effect works in clinical practice. In the case of hematological malignancies, the administered anticancer agents (ACA) can physically interact with the malignant cells, making it easier to reflect in vitro data. In solid tumors, however, the extravasated ACAs must diffuse evenly within the whole tumor mass. Therefore, the cancer stroma and the tumor mass itself can be obstacles to drug delivery systems (DDS) including antibody therapeutics. We have demonstrated that hypercoagulability caused by cancer forms cancer stroma. We further showed that the more aggressive the cancer, the greater the deposition of insoluble fibrin (IF) in cancer tissue. In this background, we decided to create monoclonal antibody (mAb) that specifically binds to IF. After a long effort, a new and unique IF-specific mAb was developed. Subsequently, anti-IF mAb conjugated with an ACA using a V-L-K linker which can be cut by plasmin. Because plasmin is activated only during IF formation, the ACA is released from the ADC only when the conjugate is bound to the IF. The released ACA may readily get to cancer cells through the stromal obstacle because of its small size. The ACA also damages the capillary that nourish cancer cells. We have named this strategy cancer (CA) stroma (S) targeting (T) therapy, or CAST therapy.

Anticoagulants for Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial syndrome with a plethora of progressive, degenerative changes in the brain parenchyma, but also in the cerebrovascular and hemostatic system. A therapeutic approach for AD is reviewed, which is focused on the role of amyloid-ß protein (Aß) and fibrin in triggering intra-brain vascular dysfunction and connected, cognitive decline. It is proposed that direct oral anticoagulants (DOACs) counteract Aß-induced pathological alterations in cerebral blood vessels early in AD, a condition, known as cerebral amyloid angiopathy (CAA). By inhibiting thrombin for fibrin formation, anticoagulants can prevent accumulations of proinflammatory thrombin and fibrin, and deposition of degradation-resistant, Aß-containing fibrin clots. These fibrin-Aß clots are found in brain parenchyma between neuron cells, and in and around cerebral blood vessels in areas of CAA, leading to decreased cerebral blood flow. Consequently, anticoagulant treatment could reduce hypoperfusion and restricted supply of brain tissue with oxygen and nutrients. Concomitantly, hypoperfusion-enhanced neurodegenerative processes, such as progressive Aß accumulation via synthesis and reduced perivascular clearance, neuroinflammation, and synapse and neuron cell loss, could be mitigated. Given full cerebral perfusion and reduced Aß- and fibrin-accumulating and inflammatory milieu, anticoagulants could be able to decrease vascular-driven progression in neurodegenerative and cognitive changes, present in AD, when treated early, therapeutically, or prophylactically.

Rivaroxaban Promotes Reduction of Embolus Size within Cerebrocortical Microvessels in a Mouse Model of Embolic Stroke.

Previous reports have suggested that direct oral anticoagulants exert a prothrombolytic effect against intracardiac thrombi. We hypothesized that these anticoagulants may also help recanalize occluded intracranial arteries via prothrombolytic effects. In this study, we evaluated the effects of rivaroxaban, a direct oral anticoagulant, on fibrin emboli within the cerebrocortical microvessels in a mouse model of embolic stroke. Fibrin emboli prepared ex vivo were injected into the common carotid artery of male C57BL/6 mice, and embolization in the microvessels on the brain surface was observed through a cranial window. Oral administration of rivaroxaban was initiated a week before injection of the emboli. The number and sizes of the emboli were measured at two time points: immediately after and 3 h after the embolus injection in the rivaroxaban-treated mice (n =6) and untreated mice (n =7). The rates of recanalization and change in the embolus size were analyzed between the two groups. Complete recanalization was observed only in the rivaroxaban group (three mice in the rivaroxaban group compared with none in the control group). A significantly higher rate of reduction of the embolus size was observed in the rivaroxaban group than in the control group (P=0.0216). No significant differences between the two groups were observed in the serum levels of the following coagulation markers: thrombin-antithrombin III complexes, D-dimers, or plasmin-α2-plasmin inhibitor complex. Our findings indicate that rivaroxaban may promote reduction in the size of stagnated fibrin emboli in cerebrocortical microvessels in cases of embolic stroke.

Inhibition of tyrosine kinase activity prevents the adhesive and cohesive properties of platelets and the expression of procoagulant activity in response to collagen.

INTRODUCTION: Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role. MATERIAL AND METHODS: Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation. RESULTS: Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced. CONCLUSIONS: Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined. MATERIALS AND METHODS: Four types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts. RESULTS: Terpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line. CONCLUSION: In addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation.

Chemotherapy payload of anti-insoluble fibrin antibody-drug conjugate is released specifically upon binding to fibrin.

Cancer-induced blood coagulation in human tumour generates insoluble fibrin (IF)-rich cancer stroma in which uneven monoclonal antibody (mAb) distribution reduce the potential effectiveness of mAb-mediated treatments. Previously, we developed a mAb that reacts only with IF and not with fibrinogen (FNG) or the fibrin degradation product (FDP). Although IF, FNG and FDP share same amino acid sequences, the mAb is hardly neutralised by FNG and FDP in circulation and accumulates in fibrin clots within tumour tissue. Here, we created an antibody drug conjugate (ADC) using the anti-IF mAb conjugated with a chemotherapy payload (IF-ADC). The conjugate contains a linker severed specifically by plasmin (PLM), which is activated only on binding to IF. Imaging mass spectrometry showed the substantial intratumour distribution of the payload following the IF-ADC injection into mice bearing IF-rich 5-11 xenografts derived from pancreatic tumours of LSL-KrasG12D/+; LSL-Trp53R172H/+; Ptf1a-Cre (KPC) mice. IF-ADC treatment significantly extended the survival of the KPC mice. These data suggest that conjugating chemotherapy drugs to this IF-specific mAb could represent an effective means of treating stroma-rich tumours.

A pathological inhibitor of fibrin cross-linking.

Lewis et al. recently reported on a patient who died of hemorrhages attributable to an acquired inhibitor of fibrin-stabilizing factor. They indicated that the inhibitor was associated with the immune globulins. Using the postmortem serum in the isolated fibrin cross-linking system, we have now further localized the site of inhibition in the scheme of blood coagulation. The interference occurs at the transpeptidation step catalyzed by the thrombin-activated fibrin-stabilizing factor. The patient's serum also uniquely delayed the clotting time of Homarus plasma, a test for specific inhibitors of transpeptidation. Since the inhibitor was effective in two such widely different systems, it probably is not an antibody, but falls into the category of cross-linking inhibitors which we have previously described (4, 5, 10, 12-17). While the exact nature of the inhibitor remains unknown, we raise the question whether some unusual metabolic transformation of isonicotinic acid hydrazide (with which the patient was treated and which itself we found to be a potent inhibitor fibrin cross-linking), in combination with a macromolecule, might not have given rise to an inhibitory compound.

Platelet interaction with polymerizing fibrin.

Interaction of washed pig, rabbit, or human platelets with fibrinogen was studied during its transition to fibrin using photometric, isotopic, and electron microscopic techniques. Untreated fibrinogen and fully polymerized fibrin had no detectable effect on platelets. Fibrinogen, incubated with low concentrations of reptilase or thrombin, formed intermediate products which readily became associated with platelets and caused their aggregation. Neutralization of the thrombin did not prevent this interaction. In the absence of fibrinogen, reptilase did not affect platelets. The interaction of polymerizing fibrin with platelets was accompanied by small losses of platelet constituents (serotonin, adenine nucleotides, platelet factor 4, and lactic dehydrogenase). This loss did not appear to be the result of the platelet release reaction. Inhibitors of the release reaction or of adenosine diphosphate (ADP)-induced aggregation did not prevent the interaction of platelets with polymerizing fibrin. Apyrase or prostaglandin E(1) (PGE(1)) reduced the extent of platelet aggregation by polymerizing fibrin, but the amount of protein associated with platelets was slightly increased. The interaction of polymerizing fibrin with platelets was completely inhibited by ethylenediaminetetraacetate (EDTA) or ethylene glycol bis (beta-aminoethyl ether) N, N,N',N'-tetraacetic acid (EGTA).Fibers formed in solutions of polymerizing fibrin were larger in the presence than in the absence of washed platelets, suggesting that platelets affect fibrin polymerization. The adherence of platelets to polymerizing fibrin may be responsible for the establishment of links between platelets and fibrin in hemostatic plugs and thrombi.

The regulation by fibrinogen and fibrin of tissue plasminogen activator kinetics and inhibition by plasminogen activator inhibitor 1.

BACKGROUND: Tissue plasminogen activator (tPA) is unusual in the coagulation and fibrinolysis cascades in that it is produced as an active single-chain enzyme (sctPA) rather than a zymogen. Two chain tPA (tctPA) is produced by plasmin but there are conflicting reports in the literature on the behaviour of sc- and tctPA and little work on inhibition by the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) under physiological conditions. OBJECTIVES: To perform a systematic study on the kinetics of sctPA and tctPA as plasminogen activators and targets for PAI-1. METHODS: Detailed kinetic studies were performed in solution and in the presence of template stimulators, fibrinogen and fibrin, including native fibrin and partially digested fibrin. Numerical simulation techniques were utilized to cope with the challenges of investigating kinetics of activation and inhibition in the presence of fibrin(ogen). RESULTS: Enzyme efficiency (k(cat)/K(m)) was higher for tctPA than sctPA in solution with chromogenic substrate (3-fold) and plasminogen (7-fold) but in the presence of templates, such as fibrinogen and native or cleaved fibrin, the difference disappeared. sctPA was more susceptible to PAI-1 in buffer solution and in the presence of fibrinogen; however, in the presence of fibrin, PAI-1 inhibited more slowly and there was no difference between sc and tctPA. CONCLUSIONS: Fibrinogen and fibrin modulate the activity of tPA differently in regard to their activation of plasminogen and inhibition by PAI-1. Fibrinogen and fibrin stimulate tPA activity against plasminogen but fibrin protects tPA from PAI-1 to promote fibrinolysis.