Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation

Abstract

Background: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate myosin light chain kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation.

Methods and results: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5-fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77-fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a-deficient embryos.

Conclusion: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.

Figure 1. Laser induced thrombosis in Tg(cd41:GFP) zebrafish larvae (5 dpf) and the effect of the knockdown of prothrombin

(A) Endothelial cells in the pericardinal vein were damaged by laser irradiation. Approximately five seconds after endothelial injury the first blood cells adhered to the vessel wall and started forming a thrombus (Ai), which grew rapidly within the next seconds (Aii). (B) To keep variation between fish as low as possible the same point (5 somites posterior to the anal fin) was taken as a target for laser irradiation of the endothelium. (C-G) Induction of a thrombus in Tg(cd41:GFP) resulted in fluorescent cells adhering to the injured vessel wall. These cells were identified as zebrafish thrombocytes. The arrowhead indicates the location of laser injury. Panels D-G display different intervals after laser injury. (H) The average time to occlusion after knockdown of prothrombin by an ATG-morpholino (MO_prothrombin) compared to uninjected controls (UIC). A significant difference is indicated by *.

Figure 2. Expression of mlck genes in thrombocytes

(A) A single cells suspension of about thousand 3 day old Tg(cd41:GFP) zebrafish embryos was sorted on GFP expression by FACS. Of the total cell population (Ai) 0.18% of the cells showed positive GFP expression (Aii). Four thousand GFP⁺ cells (purity of 83.8% after two rounds of sorting) were sorted from these embryos and compared to 4000 GFP⁻ cells (Aiii) by microarray analysis. (B) Micro-array analysis of RNA of four thousand GFP⁺ cells compared to four thousand GFP⁻ cells. Cd41 was used as a positive control. The data show 4.5 times higher expression of mlck1a (no probes were available for mlck2 and mlck3).

Figure 3. In situ hybridization with mlck specific probes after induction of a thrombus in the pericardinal vein

(A) After induction of a thrombus, embryos were fixed and wholemount in situ hybridization was performed with probes specific for the thrombocyte marker cd41 and mlck1a. The probe for cd41 was used as a positive control and showed staining of single cells on the site of thrombus formation. Sections of these embryos showed staining at the location of the pericardinal vein. The probe for mlck1a clearly displayed a similar staining pattern as the cd41 probe on wholemount and sectioned embryos. (B) Probes for mlck1b, mlck2, and mlck3 showed no staining at the site of thrombus formation.

Figure 4. Knockdown of mlck1a attenuates spreading of cd41-GFP⁺ cells on fibrinogen

(A) Target sites of the 2 splice site morpholinos (MO1_mlck1a and MO2_mlck1a) for mlck1a. Both were designed to target an exon-intron boundary upstream of the 5′ end of the protein kinase domain. (B) Expression by RT-PCR of mlck1a in the embryos injected with a p53 targeting morpholino showed a normal sized band. Injection of MO1 resulted in two bands, a wildtype band and a shorter band. Injection of MO2 resulted in three bands: a wildtype band and two shorter bands. (C-D) Fluorescent thrombocytes on fibrinogen coated slides at room temperature. Images of fluorescent channel of the different morphologic features of spreading cd41-GFP⁺ cells on fibrinogen. After 30 minutes, mainly two different cells morphologies were seen: (C) round cells, absence of initiation of cells shape change (D) cells that were fully spread on fibrinogen. (E) Number of thrombocytes that preserved their round morphology in knockdowns of mlck1a by MO1_mlck1a and MO2_mlck1a compared to the control situation (MO_p53).

Figure 5. Knockdown of mlck1a increases the time to occlusion

Time to occlusion was measured in knockdown of mlck1a by two (MO1_mlck1a and MO2_mlck1a) morpolinos and in controls (MO_p53). Time to occlusion was defined as the time between the start of laser irradiation and complete arrest of blood flow.